We are grateful to our laboratory members and the Kennedy Institute of Rheumatology community for constructive scientific discussions, especially our flow cytometry facility manager Jonathan Webber. This work was funded by Wellcome Trust Principal Research Fellowship 100262Z/12/Z, the ERC Advanced Grant (SYNECT AdG 670930), and the Kennedy Trust for Rheumatology Research (KTRR) (all three to MLD). PFCD was supported by EMBO Long-Term Fellowship (ALTF 1420-2015, in conjunction with the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Sklodowska-Curie Actions) and an Oxford-Bristol Myers Squibb Fellowship.

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The authors have no conflict of interest to declare.

BSLBs are versatile tools for studying the particulate output of T cells stimulated with model APC membranes. The flexibility of the method allows the reconstitution of complex and reductionist membrane compositions to study the effects of ligands and their signals on the secretion of tSVs and supramolecular attack particles and their components. We have tested this technology on various T cells, including preactivated TH, CTL, Tregs, and CART15. This protocol also works for the measurement of synaptic particle release of freshly isolated and quiescent T cells. One limitation of using freshly isolated T cells is that these quiescent populations produce a different profile of trans-synaptic particles, which correlates with their cell surface composition (see 15 for more details).
As a simple flow cytometry panel, we recommend the use of anti-TCR clone IP26, anti-CD81 clone 5A6, anti-perforin (PRF1) clone B-D48 or anti-CD40L clone 24-31, and ATTO 390 or ATTO 565-containing lipids (to confer BSLBs an intrinsic fluorescence). To help discriminate single cells from single BSLBs and conjugates, we recommend the use of anti-CD4 clone OKT4 and/or anti-CD45 clone HI30, which are transferred to BSLBs at limited levels despite being expressed at very high levels at the cell surface (see Table of Materials for further details on fluorochromes and other validated antibodies). Panels with a higher number of fluorochromes can be designed but require a systematic evaluation of the fluorescence spectrum spillover of each fluorochrome analyzed. To increase sensitivity, try different titrations of quantification antibodies ranging from 0.5 &#181;g to 20 &#181;g/mL final, and repeat whenever new stocks of antibodies are used. To ensure reproducibility of the absolute and relative measurements of particle transfer, titrate and calculate the binding capacity of each new lot of biotinylated and Ni-containing phospholipids, as they might differ significantly from lot to lot. The gradual cool-down of T cell-BSLB conjugates is critical to increasing the sensitivity of detection of particles with low abundance. The rapid cool-down of cocultures leads to the destruction of BSLBs, as evidenced by the significant loss of lipid fluorescence (Figure 3D).
Different metrics can be used to measure the particulate output of T cell immune synapses depending on the experimental question at hand. For instance, when minor differences are expected in the baseline expression levels of surface proteins sorted into budding SEs, a normalized synaptic transfer (NST%) metric can be used (Figure 3C panel (ii) top equation). The latter quantifies the percent MFI signal on BSLBs as a function of the total, combined MFI of cells and beads. One caveat from this approach is the analysis of transferred markers not expressed on the PM, such as components of supramolecular attack particles19. As these elements reach BSLBs by pathways independent of PM transit, such as intracellular store exocytosis, the calculation of NST% is not recommended as the result will be inflated because the numerator will be divided by a comparatively small denominator (cell surface expression level). Instead, use corrected MFIs to compare the deposition of perforin and granzymes between null BSLBs and BSLBs presenting increasing densities of antigen or anti-CD3 Fab. Alternatively, to track intracellular elements transferred to BSLBs, use for comparison either the estimated absolute molecules transferred or the percent of signal with regard to the maximum signal transferred to BSLBs (Tmax%) (Figure 3C panel (ii) bottom equation). For Tmax%, use either cMFI or molecules measured on the BSLB sample (or condition) presenting the highest antigen density as the reference Tmax. When analyzing different donors, use donor-specific Tmax for comparisons. When studying the material transferred in response to the specific triggering of the TCR, MFIs can also be corrected to the level of background transfer observed on antigen-negative (null) BSLBs.
Estimating the absolute number of molecules transferred to BSLBs is a more robust method, as this involves MESF calibration with the measurement of molecules as the endpoint and a better comparison of independent experiments. Tmax% offers a similar normalization across independent experiments and is particularly useful when using polychromatic FCM for intracellular markers, such as perforin and granzymes, or for markers for which no MESF standard is available. Tmax% is simply the percentage of signal in any given condition to the condition with the highest transfer in the control condition (e.g., vehicle/untreated controls for drug studies, control guide RNAs for CRISPR/Cas9 libraries). Further, Tmax% can be used for both cMFIs and an absolute number of molecules and has been particularly useful for the side-by-side comparisons of the effects of gene deletions on the synaptic output of T cells. The latter is evident when gene-editing leads to high variability in the dynamic range of immune receptor expression among independent donors and experiments, which might impact absolute and NST% measurements.
BSLBs have facilitated the capture and characterization of the synaptic output of different T cell types, which otherwise are difficult to isolate due to their rapid internalization by APCs2. The physical stability of BSLBs also provides a platform for the fluorescence-activated cell sorting of BSLBs that have been surveyed by T cells, thus enabling the biochemical characterization of highly pure particle preparations by mass spectrometry and nucleic acid sequencing technologies. The latter facilitates the detailed characterization of intercellular messengers shed by T cells under a broad range of experimental conditions. Different questions can be addressed, including how these particulate messengers change among different T cell types and activation states, how canonical and noncanonical antigen receptors (i.e., chimeric antigen receptors), and how agonistic and antagonistic membrane signals influence particle composition. We are currently developing this technology further for the quantitative characterization of cytokines secreted at the immune synapse of stimulated T cells. The latter requires the careful study of combinations of recombinant cytokine receptors and antibodies, providing efficient capture of interleukins, interferons, and chemokines deposited on BSLBs following T cell activation. BSLBs can be adapted to model the surface composition of other APCs suspected to trigger tSV release by T cells, such as stromal and innate immune cells.&#160;BSLBs can also be adapted to screen new pharmacological compounds seeking the positive and negative modulation of the exocytic and tSV secretion machinery for the treatment of cancer and other pathologies. Finally, BSLBs can also be used to discover virulence determinants modulating T cell function in infectious diseases20.

The immunological synapse (IS) is a pivotal molecular structure formed at the interface of cells engaged in physical contact that facilitates the regulated exchange of juxtracrine information. Different ISs have been described in the literature, and a growing body of evidence suggests these molecular hubs are a conserved feature of cellular networks. Various immune cells, including B cells, natural killer cells, dendritic cells, macrophages, and T cells, exchange information via the assembly of short-lived contacts1. Multiomic studies are advancing the understanding of novel subsets of leukocytes and stromal cells driving pathogenic cellular networks and expressing surface proteins with unknown functions. As synthetic APCs, BSLBs allow the direct investigation of the functional role of individual proteins in the integration of activating signals, namely antigens and costimulation/corepression, by T cells and the resulting release of effector particles referred to as signal four.
This paper describes the protocols and critical technical points to consider while using BSLBs to mimic the surface composition of model APCs. The protocols for the quantitative measurement of immune receptors and other surface proteins on APCs are presented along with the protocol for the reconstitution of synthetic APCs containing these measured quantities. Then, the steps required for coculturing T cells and BSLB are presented along with the protocol for the quantitative measurement of trans-synaptic particle transfer using flow cytometry. Most remarkably, BSLBs facilitate studying a plasma membrane-derived population of tSVs termed synaptic ectosomes (SEs). T-cell antigen receptor-enriched (TCR+) SEs are shed in response to TCR triggering2 and efficiently captured by BSLBs3, representing an excellent readout to assess the agonistic properties of antigens and the modelled membrane composition. CD63+ exosomes and supramolecular attack particles (SMAPs) are also released by stimulated T cells and captured by BSLBs. They can be used as additional readouts of activation and the resulting exocytic and lytic granule secretion by T cells. The mobilization of exocytic vesicles to the interacting pole of the T cell also facilitates the directional release of cytokines, such as IL-2, IFN-&#947;, and IL-10 in response to activation4,5,6,7,8. Although T-cell released cytokines can also be detected on BSLBs, a more dedicated study is currently under development to validate the quantitative analysis of cytokine release at the immunological synapse.
To interrogate how specific membrane compositions influence T cells&#39; synaptic output requires defining the physiological density of the target membrane component. Flow cytometry-based quantifications of cell surface proteins are an essential step in this protocol and require: 1) the use of antibodies with known numbers of fluorochromes per antibody (F/P), and 2) benchmark beads providing a standard reference for interpolating fluorochrome molecules from measured mean fluorescence intensities (MFIs).
These benchmark standards consist of five bead populations, each containing an increasing number of equivalent soluble fluorochromes (MESFs), which span the dynamic range of arbitrary fluorescence detection. These standard populations yield discrete fluorescence peaks, facilitating the conversion of arbitrary fluorescence units into MESFs by simple linear regression. The resulting MESFs are then used alongside antibody F/P values to calculate the average number of bound molecules per cell (or BSLB in later steps). The application of estimated cell surface areas to the average number of detected molecules then enables the calculation of physiological densities as molecules/&#181;m2. This quantification protocol can also be adapted to the measurement of protein densities on T cells and the biochemical reconstitution of membrane compositions mediating the formation of homotypic T cell synapses (i.e., T-T synapses9). If needed, the valency of antibody binding can be further estimated by using recombinant targets labeled with known numbers of fluorochromes per molecule. Then, the antibody-binding valency can be calculated for the same BSLB population by simultaneously comparing the number of bound fluorescent proteins and quantification antibodies (using two different quantification fluorochromes and MESF standards).
The reconstitution of APC membranes requires the assembly of supported lipid bilayers (SLBs) on silica beads1. Liposome stocks containing different phospholipid species can be harnessed to form a versatile lipid-bilayer matrix, enabling the anchoring of recombinant proteins with different binding chemistry (the preparation of liposomes is detailed in 10). Once the physiological density (or densities) of the relevant ligand &#34;on cells&#34; is defined, the same flow cytometry protocol is adapted to estimate the concentration of recombinant protein needed to coat BSLBs with the target physiological density. Two different anchoring systems can be used either in combination or separately.
First, SLB containing a final 12.5 mol% of Ni2+-containing phospholipids is sufficient to provide approximately 10,000 His-tag binding sites per square micron10 and works well to decorate BSLBs with most commercially available proteins whose physiological densities do not exceed this maximum loading capacity. The second loading system harnesses biotin-containing phospholipids (as mol%) to load biotinylated anti-CD3e Fab (or HLA/MHC monomers) via streptavidin bridges. The combination of these two BSLB decoration methods then enables the flexible tailoring of BSLBs as synthetic APCs. For highly complex APC surface compositions, the mol% of phospholipids and proteins can be increased to load as many proteins as the question at hand requires. Once the working concentrations of proteins and mol% of biotinylated phospholipids are defined, BSLBs can be assembled to interrogate the synaptic output of T cells with multiparametric flow cytometry.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)</td><td>Avanti Polar Lipids</td><td>790404C-25mg</td><td>18:1 DGS-NTA(Ni) in chloroform</td></tr><tr><td>&lt;br/&gt; PIPETMAN L Multichannel P8x200L, 20-200 &amp;micro;L</td><td>Gilson</td><td>FA10011</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850375C-25mg</td><td>&amp;nbsp;18:1 (&amp;Delta;9-Cis) PC (DOPC) in chloroform</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 390</td><td>ATTO-TEC</td><td>AD 390-165</td><td>DOPE ATTO 390</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 488</td><td>ATTO-TEC</td><td>AD 488-165</td><td>DOPE ATTO 488</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 565</td><td>ATTO-TEC</td><td>AD 565-165</td><td>DOPE ATTO 565</td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)</td><td>Avanti Polar Lipids</td><td>870273C-25mg</td><td>18:1 Biotinyl Cap PE in chloroform</td></tr><tr><td>200 &amp;micro;L yellow tips 10 x 96 Tips, Stack</td><td>Starlab</td><td>S1111-0206</td><td></td></tr><tr><td>5 mL polystyrene round-bottom tubes</td><td>Falcon&amp;reg;</td><td>352052</td><td></td></tr><tr><td>5.00 &amp;plusmn; 0.05 &amp;micro;m non-functionalized silica beads</td><td>Bangs Laboratories Inc.</td><td>SS05003</td><td></td></tr><tr><td>96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, non-pyrogenic.</td><td>Costar&amp;reg;</td><td>3799</td><td>For FCM staining and co-culture of BSLB and cells.</td></tr><tr><td>96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, non-pyrogenic.</td><td>Costar&amp;reg;</td><td>3894</td><td>For FCM staining of cells or beads in suspension.</td></tr><tr><td>Alexa Fluor 488 NHS Ester (Succinimidyl Ester)</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>A20000</td><td></td></tr><tr><td>Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>A37573 and A20006</td><td></td></tr><tr><td>Allegra X-12R Centrifuge</td><td>Beckman Coulter</td><td></td><td>For normal in tube staining of biological samples for FCM</td></tr><tr><td>Aluminum Foil</td><td>Any brand</td><td></td><td>For protecting cells and BSLBs from light</td></tr><tr><td>anti-human CD154 (CD40L), clone 24-31</td><td>BioLegend</td><td>310815 and 310818</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>anti-human CD185 (CXCR5) Brilliant Violet 711, clone J252D4</td><td>BioLegend</td><td>356934</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD19 Brilliant Violet 421, clone HIB19</td><td>BioLegend</td><td>302234</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD2, clone RPA-2.10</td><td>BioLegend</td><td>300202</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD2, clone TS1/8</td><td>BioLegend</td><td>309218</td><td>Brilliant Violet 421 conjugate.</td></tr><tr><td>anti-human CD252 (OX40L), clone 11C3.1</td><td>BioLegend</td><td></td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD28, clone CD28.2</td><td>eBioscience</td><td>16-0289-85</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD317 (BST2, PDCA-1), clone 26F8</td><td>ThermoFisher Scientific, invitrogen</td><td>53-3179-42</td><td>Alexa Fluor 488 conjugate, we found this clone to be cleaner than clone RS38E.</td></tr><tr><td>anti-human CD38, clone HB-7</td><td>BioLegend</td><td>356624</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human CD38, clone HIT2</td><td>BioLegend</td><td>303514</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD39, clone A1</td><td>BioLegend</td><td></td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD4 Brilliant Violet 650, clone OKT4</td><td>BioLegend</td><td>317436</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD4, clone A161A1</td><td>BioLegend</td><td>357414 and 357421</td><td>PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD4, clone OKT4</td><td>BioLegend</td><td>317414 and 317422</td><td>PE/Cy7 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD40, clone 5C3</td><td>BioLegend</td><td>334304</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD40, clone G28.5</td><td>BioLegend</td><td></td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD45, clone HI30</td><td>BioLegend</td><td>304056 and 368516</td><td>Alexa Fluor 647 and APC/Cy7 conjugates</td></tr><tr><td>anti-human CD47, clone CC2C6</td><td>BioLegend</td><td>323118</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD54 (ICAM-1), clone HCD54</td><td>BioLegend</td><td>322702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD63 (LAMP-3), clone H5C6</td><td>BioLegend</td><td>353020 and 353015</td><td>PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD73, clone AD2</td><td>BioLegend</td><td>344002</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD80, clone 2D10</td><td>BioLegend</td><td>305216</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD81, clone 5A6</td><td>BioLegend</td><td>349512 and 349502</td><td>PE/Cy7 conjugate and labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester), respectively.</td></tr><tr><td>anti-human CD82, clone ASL-24</td><td>BioLegend</td><td>342108</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD86, clone IT2.2</td><td>BioLegend</td><td>305416</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD8a, clone HIT8a</td><td>BioLegend</td><td>300920</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human CD8a, clone SK1</td><td>BioLegend</td><td>344724</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human HLA-A/B/C/E, clone w6/32</td><td>BioLegend</td><td>311414 and 311402</td><td>Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human HLA-DR, clone L243</td><td>BioLegend</td><td>307656 and 307622</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>anti-human ICAM-1, clone HCD54</td><td>BioLegend</td><td>322702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human ICOS, clone C398.4A</td><td>BioLegend</td><td>313516</td><td>Armenian Hamster IgG</td></tr><tr><td>anti-human ICOSL, clone MIH12</td><td>BioLegend</td><td>329611</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human ICOSL, clone MIH12</td><td>eBioscience</td><td>16-5889-82</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human LFA-1, clone TS1/22</td><td>BioLegend</td><td>Produced in house</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human OX40, clone Ber-ACT35 (ACT35)</td><td>BioLegend</td><td>350018</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human PD-1 , clone EH12.2H7</td><td>BioLegend</td><td>135230 and 329902</td><td>Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human PD-L1, clone 29E.2A3</td><td>BioLegend</td><td>329702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human PD-L2, clone 24F.10C12</td><td>BioLegend</td><td>329611</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human PD-L2, clone MIH18</td><td>BioLegend</td><td>345502</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human TCRab, clone IP26</td><td>BioLegend</td><td>306712 and 306714</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>antti-human CD156c (ADAM10), clone SHM14</td><td>BioLegend</td><td>352702</td><td></td></tr><tr><td>antti-human CD317 (BST2, Tetherin), clone RS38E</td><td>BioLegend</td><td>348404</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>Armenian Hamster IgG Alexa Fluor 647 Isotype control, clone HTK888</td><td>BioLegend</td><td>400902</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>BD Cytometer Setup and Tracking beads</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>641319</td><td>Performance track of instruments before quantitative FCM</td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>23-14523-00</td><td>Acquisition software</td></tr><tr><td>Bovine Seum Albumin</td><td>Merck, Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;, Calcium chloride</td><td>Merck, Sigma-Aldrich</td><td>C5670</td><td>anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;96.0%</td></tr><tr><td>Casein from bovine milk, suitable for substrate for protein kinase (after dephosphorylation), purified powder</td><td>Merck, Sigma-Aldrich</td><td>C5890</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>ThermoFisher Scientific, Gibco</td><td>11132D</td><td></td></tr><tr><td>DynaMag-2</td><td>ThermoFisher Scientific, Invitrogen&amp;trade;</td><td>12321D</td><td>For the removal of Dynabeads Human T-Activator CD3/CD28 in volumes less than 2 mL</td></tr><tr><td>DynaMag&amp;trade;-15</td><td>ThermoFisher Scientific, Invitrogen&amp;trade;</td><td>12301D</td><td>For the removal of Dynabeads&amp;trade; Human T-Activator CD3/CD28 in volumes less than 15 mL</td></tr><tr><td>Fetal Bovine Serum Qualified, One Shot</td><td>ThermoFisher Scientific, Gibco</td><td>A3160801</td><td>Needs heat inactivation for 30 min at 56 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva, GE Healthcare</td><td>GE17-1440-02</td><td>Sterile solution of polysaccharide and sodium diatrizoate for lymphocyte isolation</td></tr><tr><td>Fixable Viability Dye eFluor 780</td><td>eBiosciences</td><td>65-0865-14</td><td>For the exclusion of dead cells during analyses</td></tr><tr><td>FlowJo</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>Version 10.7.1</td><td>Analysis software</td></tr><tr><td>Grant Bio MPS-1 Multi Plate Shaker</td><td>Keison Products</td><td>MPS-1</td><td>For the mixing of either cells during stainings or BSLBs during staning or protein loading (as an alternative to orbital agitation)</td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>ThermoFisher Scientific, Gibco</td><td>15630-056</td><td></td></tr><tr><td>HEPES, N-(2-Hydroxyethyl)piperazine-N&amp;prime;-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid.</td><td>Merck, Sigma-Aldrich</td><td>H4034</td><td>For preparation of HBS/HAS or HBS/BSA buffer. BioPerformance Certified, &amp;ge;99.5% (titration), suitable for cell culture</td></tr><tr><td>HERACell 150i CO&lt;sub&gt;2&lt;/sub&gt; incubator, 150 L, Electropolished Stainless Steel</td><td>ThermoFisher Scientific</td><td>51026282</td><td>For culturing and expanding purified CD4+ and CD8+ T cells.</td></tr><tr><td>Hula Mixer&amp;reg; Sample Mixer</td><td>ThermoFisher Scientific, Life Technologies</td><td>15920D</td><td>Vertical, variable-angle laboratory mixer used for the mixing of BSLBs and lipid master mix, blocking solutions, protein master mix and small scale antibody stainings.</td></tr><tr><td>Human Serum Albumin, 30% aqueous solution</td><td>Merck, Sigma-Aldrich</td><td>12667-M</td><td></td></tr><tr><td>Human TruStain FcX Fc Receptor Blocking Solution</td><td>BioLegend</td><td>422302</td><td>Fc Receptor Blocking Solution for blocking of Fc Receptors from biologically relevant samples</td></tr><tr><td>Innovatis CASY cell counter and analyzer TT</td><td>Biovendis Products GmbH</td><td></td><td>For the counting of cells and the determination of cell size and volume based on the exclusion of electric current.</td></tr><tr><td>KCl, Potassium chloride</td><td>Merck, Sigma-Aldrich</td><td>P5405</td><td>Powder, BioReagent, suitable for cell culture</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher Scientific, Gibco</td><td>25030-024</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;, Magessium chloride</td><td>Merck, Sigma-Aldrich</td><td>M2393</td><td>BioReagent, suitable for cell culture, suitable for insect cell culture</td></tr><tr><td>Microtube Insert for 24 x 1.5/2.0 mL tubes</td><td>Keison Products</td><td>P-2-24</td><td>Microtube insert for Grant Bio MPS-1 Multi Plate Shaker</td></tr><tr><td>Mini Incubator</td><td>Labnet International&amp;nbsp;</td><td>I5110A-230V</td><td>For the incubation (co-culturing) of BSLB and cells in the absence of CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Minimum Essential Medium Non-Essential Amino Acids</td><td>ThermoFisher Scientific, Gibco</td><td>11140-035</td><td></td></tr><tr><td>Mouse IgG polyclonal antibody control</td><td>Merck, Sigma-Aldrich</td><td>PP54</td><td>Used as positive control for the measurement of antibodies bound to mouse IgG capture bead standards</td></tr><tr><td>Mouse IgG1, k Isotype, clone MOPC-21</td><td>BioLegend</td><td>400129, 400112, 400130, 400144, 400128 and 400170</td><td>Alexa Fluor 488, PE, Alexa Fluor 647, Alexa Fluor 700, APC/Cyanine7 and Brilliant Violet 785&amp;nbsp; conjugates, respectively.</td></tr><tr><td>Mouse IgG1, k Isotype, clone X40</td><td>Becton Dickinson &amp;amp; Company (BD), Horizon</td><td>562438</td><td>Brilliant Violet 421 conjugate.</td></tr><tr><td>Mouse IgG1, &amp;kappa; Isotype control, clone P3.6.2.8.1</td><td>eBioscience</td><td>14-4714-82</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>Mouse IgG2a, k Isotype, clone MOPC-173</td><td>BioLegend</td><td>400240</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>Mouse IgG2b, k Isotype, clone MPC-11</td><td>BioLegend</td><td>400330 and 400355</td><td>Alexa Fluor 647 and Brilliant Violet 785 conjugates, respectively</td></tr><tr><td>Multiwell 6 well Tissue culture treated with vacuum gas plasma</td><td>Falcon</td><td>353046</td><td>For culturing and expanding purified CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; T cells.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, Disodium Phosphate</td><td>Merck, Sigma-Aldrich</td><td>S7907</td><td></td></tr><tr><td>NaCl, Sodium chloride</td><td>Merck, Sigma-Aldrich</td><td>S5886</td><td>BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;99%</td></tr><tr><td>NiSO&lt;sub&gt;4&lt;/sub&gt;, Nickel(II) sulfate</td><td>Merck, Sigma-Aldrich</td><td>656895</td><td>For saturating NTA sites; added during the blocking process</td></tr><tr><td>Penicillin Streptomycin [+]10,000 units Penicillin; [+] 10,000 &amp;micro;g/mL Streptomycin</td><td>ThermoFisher Scientific, Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4, sterile</td><td>ThermoFisher Scientific, Gibco</td><td>10010</td><td>No Ca&lt;sup&gt;2+&lt;/sup&gt; or Mg&lt;sup&gt;2+&lt;/sup&gt; added</td></tr><tr><td>Polyethersulfone (PES) Filter unit</td><td>Thermo Scientific Nalgene</td><td>&amp;nbsp;UY-06730-43</td><td>Hydrophilic PES membrane with low protein binding facilitates the filtering of solutions with high protein content</td></tr><tr><td>PURESHIELD argon ISO 14175-I1-Ar</td><td>BOC Ltd.</td><td>11-Y</td><td>For the protection of lipid stocks stored at +4 &amp;ordm;C.</td></tr><tr><td>Purified Streptavidin</td><td>BioLegend</td><td>280302</td><td></td></tr><tr><td>Quantum Alexa Fluor 488 MESF beads</td><td>Bangs Laboratories Inc.</td><td>488</td><td>Benchmark beads for the interpolation of Alexa Fluor 488 molecules bound to cells and/or BSLB</td></tr><tr><td>Quantum Alexa Fluor 647 MESF beads</td><td>Bangs Laboratories Inc.</td><td>647</td><td>Benchmark beads for the interpolation of Alexa Fluor 647 molecules bound to cells and/or BSLB</td></tr><tr><td>Rat anti-mouse IgG Kappa Light Chain, clone OX-20</td><td>ThermoFisher Scientific, invitrogen</td><td>SA1-25258</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>200-02-1MG</td><td></td></tr><tr><td>RMPI Medium 1640 (1x); [-] L-Glutamine</td><td>ThermoFisher Scientific, Gibco</td><td>31870-025</td><td></td></tr><tr><td>Rosette Human B Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15064</td><td>Isolation of B cells for measuring densities of proteins in purified cell populations</td></tr><tr><td>Rosette Human CD4+ T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15022C.1</td><td></td></tr><tr><td>Rosette Human CD4+CD127low T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15361</td><td>Pre-enrichment of CD4+ CD127&lt;sup&gt;Low&lt;/sup&gt; T cells for the downstream isolation of Tregs by FACS.</td></tr><tr><td>Rosette Human CD8+ T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15063</td><td></td></tr><tr><td>RPMI Medium 1640 (1x); [-] Phenol Red</td><td>ThermoFisher Scientific, Gibco</td><td>11835-063</td><td>For the incubation (co-culturing) of BSLB and cells in the absence of CO&lt;sub&gt;2&lt;/sub&gt;. Phenol red-free media reduces the autofluorescence of cells in flow cytometry and microscopy based measurements.</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>ThermoFisher Scientific, Gibco</td><td>11360-070</td><td></td></tr><tr><td>Sprout mini centrifuge</td><td>FisherScientific, Heathrow Scientific LLC</td><td>120301</td><td>Benchtop microcentrifuge used to wash silica beads and BSLB in 1.5 mL Eppendorf tubes.</td></tr><tr><td>Sterile cappeed 5 mL polystyrene round-bottom tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>UltraComp eBeads Compensation Beads</td><td>ThermoFisher Scientific, invitrogen</td><td>01-2222-42</td><td></td></tr><tr><td>Zeba Spin Desalting Columns 7K MWCO</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>89882</td><td></td></tr></tbody></table>

preparation of bead-supported lipid bilayers to study the particulate output of t cell immune synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

FCM for absolute protein quantification on the cell surface 
The reconstitution of BSLBs presenting physiological densities of ligands requires the estimation of total protein densities on the modeled cell subset. To reconstitute BSLBs, include any relevant ligand expected to play a role in the signaling axis of interest alongside proteins supporting the adhesion and functional interaction between BSLB and cells, such as ICAM-1 and costimulatory molecules, e.g., CD40, CD58, and B7 receptors (CD80 and CD86). Additional proteins can be added depending on the question at hand, including costimulatory molecules such as ICOSL3, PD-L1, and PD-L215. For any other molecules, reconstitute BSLBs using molecular densities determined by directly analyzing cells with quantitative flow cytometry. For directly conjugated antibodies, BioLegend provides F/P values for each antibody lot number. Antibodies can also be labeled in-house and the F/P ratios determined by spectrophotometry, providing an alternative when there are no commercial antibodies conjugated to the desired fluorochrome. Since we use the same antibodies to calibrate the number of recombinant proteins on the surface of cells and BSLBs, there is no need to correct the antibody-binding valency as this remains constant. If the antibody-binding valency is required, use both recombinant proteins and antibodies with known F/P to decorate BSLBs and compare molecules of loaded recombinant proteins with the number of bound antibodies following staining under saturating conditions.
The bound antibody molecules per cell can be estimated using a dedicated monochromatic flow cytometric measurement or a polychromatic panel of antibodies intended to estimate absolute protein densities on a relatively infrequent subset of cells within a tissue of interest, such as palatine tonsils. Figure 1 shows representative measurements of densities of ICAM-1 in CXCR5+ B cells and follicular T cells (TFH) as an example. The same staining protocol and flow cytometry analysis principles shown in Figure 1A can be used to measure protein densities on epithelial cells, stromal cells, monocytes, monocyte-derived dendritic cells (moDCs), or on B and T lymphocytes in other human and mouse tissues. For tissues different from blood and tonsils, caution must be taken when isolating cells using protease cocktails, as the prolonged exposure of cells to digesting enzymes reduces cell surface expression levels.
Focus the acquisition and analyses on single, live cells within the continuous acquisition window (Figure 1A ii), as events outside the time continuum aberrantly scatter light, compromising quantification. To increase the accuracy of determinations, reduce the nonspecific staining of APCs by efficient blocking of Fc&#947;Rs. The human serum and EDTA present in hFCB enable efficient Fc&#947;R blocking while chelating free Ca2+ to reduce the spontaneous aggregation of cells during their manipulation and staining (black arrows in Figure 1A (iii) and (iv) show remaining doublets in suspensions of tonsillar cells).
Keeping track of the instrument performance by using the setup and tracking beads and the software (or similar; see the Table of Materials) is critical for the reproducibility of quantifications over time, especially in later steps when the synaptic transfer of particles to BSLB is measured using only MFIs (i.e., for fluorochromes for which there are no MESF standards). Similarly, check the linear range (i.e., linearity minimum and linearity maximum) of arbitrary fluorescence units for the quantification detector to be used alongside the MESF standards such that each calibration point keeps the linear relationship between fluorescence and the number of fluorochromes.
The preparation of samples &#34;blank&#34; for fluorescence, or samples providing an idea of the background staining noise, are essential to subtract the nonspecific fluorescent signal. Isotype controls and/or biologically null samples (e.g., knockouts) are essential to correct for the background signal of cells and extract the true signal derived from the quantification antibodies (Figure 1A panel (viii)). Similarly, standard MESF beads use a dedicated blank population to subtract the background signal from each truly positive bead population (Figure 1B panels (vii) and (viii)). Once the regression analyses are performed and the slope defining the relationship between MESF and corrected MFIs is extracted, the conversion to absolute molecular densities follows simple mathematical operations (Figure 1C).
To estimate CSA in Figure 1C, electrical current exclusion (CASY-TT) was used to extract measurements of cell volume and diameter from thousands of cells. The resulting CSA estimated from the calculation of surface area for spheres (4pr2) varies with the activation state of the cells, with observed values of 170.37 &#177; 4.91 &#181;m2 for nonactivated B cells and 234.52 &#177; 1.53 &#181;m2 and 318 &#177; 24.45 &#181;m2 for nonactivated and activated T cells, respectively. These CSAs are comparable to those estimated by imaging techniques such as three-dimensional refractive index tomography of nonactivated lymphocytes16.
Once the range of physiological densities has been defined (e.g., by comparing surface densities on cells undergoing different activation programs), BSLBs can be used to model those surfaces. A titration of biotinylated antigenic HLA-peptide monomers provided by the NIH tetramer facility (or monobiotinylated monomeric anti-CD3&#949;-Fab) can be used alongside ICAM-1 12-His to reconstitute a canonical APC membrane. Commercial proteins tagged with 6, 9, 12, and 14 His can be used to decorate the surface of BSLB (see Figure 2A for examples with ICAM-1 12-His). Protein titrations together with quantitative FCM analyses provide a robust methodology to reconstitute physiological APC surfaces and test their effect on the synaptic output of different T cell subsets.
For studying the output of T cell synapses, use a 1:1 BSLB-to-T cell ratio to ensure that, on average, one cell will interact with one BSLB over the studied period. We have observed that the material transfer is proportional to the incubation time, providing a versatile platform for detecting molecules transferred in low quantities across the cell:BSLB interface. An appropriate panel design is thus critical to increasing the sensitivity and reliability of the detection of trans-synaptic material, as in the case of tSVs, the output varies between 25 and 36 vesicles/cell/20 min2,3. Test first the spectral spillover of each fluorochrome-labeled antibody and lipid. When high spillover is observed, we recommend the titration of staining antibodies and the percent of fluorescent lipids composing the BSLB, as well as PMT voltage walks to reduce the spreading error on compensated samples and enhance the signal over noise ratio, respectively (refer to12,14,17 for a dedicated introduction to the subject).
The use of quantification controls, including null BSLBs (lacking antigen or anti-CD3 Fab) and either knockout cells or isotype-labeled samples18, is essential to accurately measure the transfer of effector tSV, such as SEs, as well as supramolecular attack particles released within the synaptic cleft. Use highly abundant cell-surface proteins such as CD4, CD2, or CD45 alongside synthetic fluorescent lipids (DOPE Atto conjugates) to identify the population of single cells and BSLB upon cold-based dissociation of conjugates. Focus the analyses on the geometric mean or median of fluorescence intensities in single BSLB and cells (CD4 is used in Figure 3B). SEs are a specialized type of tSV derived from the plasma membrane (PM), and their transfer to BSLB is evidenced by the gain of marker signal on BSLBs with the consistent loss of signal on the surface of the interacting cells (refer to TCR on BSLBs as shown in Figure 2B, violet arrows). Null BSLBs lacking either antigen or anti-CD3 are an excellent reference to keep track of the specific gaining of TCR (and other T cell markers) on BSLB resulting from stimulating interacting cells via their TCR complex.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63130/63130fig01.jpg" /> 
Figure 1: Absolute quantification of proteins on the surface of APCs. (A) Example of quantitative flow cytometry measurements of ICAM-1 on the surface of tonsillar B cells (Foll. Bc) and helper T cells (TFH). (i-vii) Gating strategy for analyzing single CXCR5+ Bc and TFH isolated from human palatine tonsils. Shown is the sequential gating strategy for identifying single, live events contained within the continuous window of acquisition. (iii-iv) black arrows indicate doublets. (viii) overlaid histograms showing the cell surface expression of ICAM-1 (teal histograms) compared to FMO controls (grey histograms) and FMO controls labeled with relevant isotypes (black histograms, which overlap with the grey histograms) of the populations shown in (vii). Arrows indicate the direction for the nested gating strategy used to identify CXCR5+ B cells (Bc; CD19+) and TFH (CD4+). (B) Extraction of absolute molecules on the surface of tonsillar cells from MFI requires regression analyses of MESF benchmark beads acquired using the same instrument setting as the cells shown in A. (i-v) Shown is the sequential gating strategy for identifying single, live events contained within the continuous window of acquisition. (vi) Gating and measurement of MFIs from different standard MESF populations. (vii) shown are overlaid histograms of the MESF populations identified in (vi). The values displayed on the top right represent the MFIs for each of the 5 MESF populations (blank, 1, 2, 3, and 4). (viii) Linear regression of MESF over cMFI for the MESF populations shown in (vii). Shown is the slope (b) for extracting MESF bound to cells from data in A. (C) In the extraction of the number of molecules, follow simple mathematical operations starting with the application of the slope calculated in (viii) from measured MESF cMFI (cMFIM) and reference MESF values (MESFR). To extract the MESF bound to cells (MESFcells), divide the corrected MFI of cells (cMFIcells) by the calculated slope. Then, to calculate the number of molecules bound to cells (Molec.cells), divide MESFcells&#160;by the F/P of the detection (quantification) antibody. Finally, to calculate the molecular density on the surface of cells (Dcells), divide Molec.cells by the estimated cell surface area (CSAE). Abbreviations: X = independent variable; Y = dependent variable (measured fluorescence), cMFIM = measured corrected MFI; MESFR = reference MESF values; MESFcells = estimated MESF per cell; cMFIcells = corrected MFI cells; Molec.cells = estimated molecules per cell. Dcells = estimated density on cells; CSAE = estimated Cell Surface Area. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63130/63130fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63130/63130fig02.jpg" /> 
Figure 2: Reconstitution of BSLB with recombinant ICAM-1 and the measurement of particulate transfer to BSLBs. (A, i-vi) Flow cytometry analysis of BSLBs reconstituted with increased densities of recombinant monomeric ICAM-1 12-His (rICAM-1). (i-v) As in Figure 1, focus the gating strategy on single BSLBs within the continuous window of acquisition. Note the gap immediately before the time continuum gate, which was excluded to prevent errors of measurement. (vi) Good protein quality often results in the homogeneous coating of BSLB at high concentrations, with the observation of narrow fluorescence distributions (low Coefficient of Variation, see histograms in vi). (B) Regression analyses of ICAM-1 reference concentration (CR) over measured density (DM). Use the slope to calculate target concentrations of protein (CT) to achieve the density of cells (Dcells) measured in the experiments in Figure 1. Abbreviations: 12-His = 12-histidines tag; DM = measured molecular densities; CR = reference concentrations of the rICAM-1; CT = target concentration (to be interpolated); Dcells = densities measured in cells (see also Fig. 1C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63130/63130fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63130/63130fig03.jpg" /> 
Figure 3: Measurement of T cell synaptic particles transferred to BSLBs. (A; i-v)&#160;Flow diagram showing the critical steps for the co-culturing of T cells with BSLBs reconstituting model membranes and the subsequent measurementof particle transfer with flow cytometry. (iv) Blue and dark yellow diagrams show the relative distribution and location of cells and BSLBs in biparametric flow cytometry plots. (v) Fluorescence distribution histograms displaying the relative gain of fluorescence of agonistic BSLBs (dark yellow) compared to null BSLBs (grey). (B)&#160;Exemplary synaptic transfer experiment. (i-vi)&#160;Shown is the gating strategy to identify single BSLBs and cells within the continuous acquisition window. Violet arrows indicate the direction of analysis, which continues in C. (C)&#160;(i)&#160;Focus the analyses on the MFI of single cells (blue) and single BSLBs (yellow). (ii) Equations to calculate the normalized synaptic transfer (NST%, top) and Tmax% (bottom) from the cMFI calculated for BSLB and cells. (iii-vi) Overlaid histograms showing the change of fluorescence intensity distributions for cells (blue shades) and BSLBs (yellow shades) across different densities of the T-cell activating anti-CD3&#949;-Fab, including non-activating (grey) and activating with either 250 (soft color value) or 1,000 (high color value) molec./&#181;m2. Numbers in different color values represent the NST% measured for the BSLB histograms shown in yellow. The overlaid histograms show the overarching hierarchy in the synaptic transfer of T cell vesicles positive for different markers. For this composition of BSLBs (200 molec./&#181;m2 of ICAM-1 and increasing densities of anti-CD3&#949;-Fab), tSVs are transferred to BSLB with TCR+(iii) &#62; CD81+(iv) &#62; CD4+(v) &#62; CD28+(vi). As demonstrated in previous articles, TCR and CD81 are components of SEs and are transferred with comparatively higher efficiencies to CD4, despite the latter being expressed at comparatively higher surface levels. SE shedding results in the loss of cell surface CD81 and TCR and the gain of these signals on BSLBs (open purple arrows for 250 molec./&#181;m2, and closed purple arrows for 1,000 molec./&#181;m2 in yellow histograms). (D) Improper cooling down of conjugates leads to cells ripping off the SLB from silica beads as seen from comparing input beads (left biparametric plot) and conjugates subjected to rapid cooling down to 4 &#176;C from 37 &#176;C (right biparametric plot). Compare also with Figure 3B panel (vi). Abbreviations: PRF1 = perforin 1; NST% = normalized synaptic transfer; Tmax% = percent of maximum observed transfer (in control or reference condition); tSVs: trans-synaptic vesicles; SEs: synaptic ectosomes. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63130/63130fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Buffers used in this protocol. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63130/Table 1_revised_63130.xls" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses at JoVE.com

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

preparation-bead-supported-lipid-bilayers-to-study-particulate-output

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

4.1K Views.  The University of Oxford. Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells. 

Video: Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biology

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengineering

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Source: Curtis, B. N. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022).
This video describes the technique of studying the septin protein assembly on microspheres coated with lipid bilayers. This technique helps in understanding the curvature-dependent assemblies of protein on curved surfaces.

Test dwuwarstwowy lipidów ze sferycznym wsparciem: technika obserwacji składania się septyny na mikrosferach

Source: Curtis, B. N. et al., Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022).
This ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells

Source: C&#233;spedes, P. F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63130">Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses.</a> J. Vis. Exp. (2022)
This video illustrates the reconstitution of synthetic antigen-presenting cells through Bead-Supported Lipid Bilayers and their application in assessing the synaptic output generated by activated T cells.

Wykorzystanie dwuwarstw lipidowych wspieranych przez kulki do badania wyjścia synaptycznego z limfocytów T

1. Protein density calibrations on BSLBs (Bead-supported lipid bilayers)

	Before taking the 5.00 &#177;0.05 &#181;m diameter non-functionalized silica ...

Immunology

Immunodiagnostics

Specialized Assays

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

To assemble the glass-supported planar lipid bilayer, first, prepare 100 milliliters of piranha solution by mixing 30% hydrogen peroxide with sulfuric acid at a ratio of 1 to 3 in a beaker. Into this solution, place two rectangular #1.5 coverslips for 20 to 30 minutes. While the coverslips are being cleaned, retrieve one tube of previously prepared 400-micromolar DOPC lipids, and one tube of previously prepared 80-micromolar Biotin-PE lipids. Transport them on ice to the argon tank.
After deoxygenating a new microcentrifuge tube with argon, add together the DOPC and Biotin-PE at a 1 to 1 ratio. Specific volume will vary based on experimental needs, but should be, at minimum, 2 microliters each. Deoxygenate the mixture tube and the individual reagent tubes before returning the latter to the refrigerator.
After they are finished cleaning, thoroughly rinse the coverslips with distilled water. Set the coverslips out to air-dry for a few minutes. Then, withdraw 1.5 microliters of the liposome mixture, and aliquot it in a single drop into one of the lane chambers of a chamber slide. The use of two drops per lane is typical but not necessary.
Now, quickly and efficiently place the dry coverslip over the droplets. Ensure that the drops are sufficiently spaced so that they do not merge once the coverslip is placed. Furthermore, make sure that the drops remain circular and well-defined, without touching the edges of the chamber walls. Press down firmly, in between and around each lane, to ensure a watertight seal between the coverslip and slide.
Mark the positions of the drops using a marker pen. Then, pass 100 microliters of aqueous 5% casein through the chamber to block the bilayer. Try to make sure that there are no bubbles in the flow chamber. Then, inject 100 microliters of streptavidin at a concentration of 333 nanograms per milliliter into each lane.
After incubating for 10 to 15 minutes at room temperature, wash by running 3 milliliters of HEPES-buffered saline with 1% human serum albumin through each lane to remove the excess streptavidin. Add 100 microliters of biotinylated, fluorescently labeled anti-CD16 at the protein concentration previously determined, as described in the text protocol.
Following incubation in the dark for 20 to 30 minutes, wash again by running 3 milliliters of HEPES-buffered saline with 1% human serum albumin through each lane. Then, flow 100 microliters of D D-biotin at a concentration 25 nanomolar through the chamber in order to bind any excess streptavidin, and thus, eliminate the chance of nonspecific binding of streptavidin to the cells.
Next, count human NK cells, and resuspend them at a concentration of 500,000 per milliliter in HEPES-buffered saline with 1% human serum albumin. Wash the D-biotin out of the chamber with another 3 milliliters of HEPES-buffered saline with 1% human serum albumin per lane. Once the cells have been resuspended at the desired concentration, add 100 microliters to each lane. Place the chamber in a 37-degree Celsius, 5% carbon dioxide incubator for 30 to 60 minutes.
After this incubation period, fix the cells with 4% paraformaldehyde at room temperature for 10 to 20 minutes. Wash by running 3 milliliters of phosphate-buffered saline through each lane to remove the paraformaldehyde. Then, add 400 microliters of blocking buffer before incubating at room temperature for 30 minutes.
Next, stain F-actin and perforin by adding 200 microliters of diluted, fluorescently labeled phalloidin and fluorescently labeled anti-perforin monoclonal antibody. Incubate at room temperature for one hour before washing by running 3 milliliters of phosphate-buffered saline through the chamber. The chamber is ready for imaging.
To begin, turn on all necessary hardware modules of the STED microscope. Start up the image analysis software, and enable both resonant scanning and STED modules. After making these selections, wait for about three to five minutes for the software to initiate.
Click on the Configuration tab at the top of the screen. Select Laser Config. Then, turn on the white light and the STED 592-nanometer lasers. Choose the 100X objective, and align the excitation laser beam with a 592-nanometer depletion laser. Select the Laser Config module. Turn off the 592 depletion laser, and turn on the 660-nanometer depletion laser.
Place the slide upon the stage over the lens. Bring the cells bound on the demarcated bilayer region into focus using the white light lamp and the oculars. Then, return to the Acquisition tab directly to the right of the Configuration tab. Click the Switch to White Light tab, and then turn that module on, and drag the excitation laser line to the appropriate wavelength.
After selecting the desired detector and setting the detection range, click on the Sequential button in the left-hand Acquire toolbar to bring up the sequential scanning dialog in the bottom of the left-hand toolbar. This allows the user to add multiple sequences, each with a different excitation beam for a different color. Click between frames, and then, set the excitation frequency, detector, and detection range for each additional color. Once all the settings are optimized, hit Start to begin the acquisition process.