We acknowledge the technical contributions of the Flow Cytometry, Microsurgery, and Bio- repository/Pathology Centers of Research Excellence at Mount Sinai. This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, Ministerio de Ciencia e Innovacion SAF2013-48834-R and SAF2016-80031-R J.O.

In this study, mice are housed in accordance with the United States Department of Agriculture guidelines and the recommendations of the Public Health Service Guide for the Care and Use of Laboratory Animals. All experimental techniques involving animal use were performed in accordance with Institutional Animal Care and Utilization Committee (IACUC)-approved protocols of the Mount Sinai School of Medicine.
1. Media Preparation
<ol>
	<li>Prepare complete RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin (10,000 U/mL) by using 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 5 mM HEPES, and 0.05 mM 2-mercaptoethanol.</li>
</ol>
2. Allograft Isolation and single-cell Suspension
NOTE: The transplantation and anastomosis technique of the pulmonary artery and inferior cava vein was initially described by Corry and collaborators and can be visualized in Liu &#38; Kang10,11 (Figure 1A).
<ol>
	<li>Anesthetize a transplanted mouse with 4-5% isoflurane in an induction chamber. Sacrifice it by cervical dislocation.</li>
	<li>Make a midline abdominal incision with standard scissors and remove the abdominal contents to localize the aorta. 
	NOTE: The transplanted heart will be on the right side of the recipient abdominal aorta (Figure 1B).</li>
	<li>Pull out the graft gently using fine sharp-teeth forceps and place it immediately into ice-cold RPMI medium. 
	NOTE: Using a surgery scissor to separate the graft from the aorta can damage the graft and cause some tissue to remain adhered to the recipient.</li>
	<li>After all grafts have been collected, move them into a sterile culture hood for tissue processing. Place the graft in a petri dish and dice the tissue into small pieces (1 mm) using sterile, blunt, microsurgery scissors.</li>
	<li>With sharp-teeth forceps, transfer the pieces to a 50 mL tube with 5 mL of collagenase A (0.1 mg/mL collagenase in sterile 1x PBS). Incubate it for 1 h in a 37 &#176;C bath.</li>
	<li>Add 5 mL of RPMI medium to neutralize the collagenase and transfer the sample through a 100 &#181;m strainer with the help of the plunger of a 1 mL syringe.</li>
	<li>Spin the sample down at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 1 mL of ACK lysis buffer. Mix well and incubate for 5 min at 4 &#176;C.</li>
	<li>Add 1 mL of RPMI medium to neutralize the lysis buffer and spin the sample down at 400 x g for 5 m at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 200 &#181;L of RPMI medium and transfer it to a 5 mL polypropylene round-bottom tube. 
	NOTE: Polypropylene tubes support less adherence. Also, maintaining the cells at low temperature, such as 4 &#176;C, reduces adhesion.</li>
</ol>
3. Isolation of Graft-infiltrating Macrophages Using Fluorescence-activated Cell Sorting
<ol>
	<li>Block unwanted specific binding on myeloid cells by using Fc receptor blocking mAb (rat anti-mouse CD16/32). Add 1-2 &#181;L per sample, 15 min prior to surface staining.</li>
	<li>Stain with anti-mouse CD11b Percp/Cy5.5 (0.6 &#181;g/&#181;L final concentration in the RPMI medium), anti-mouse CD45 APC/eFluor780 (0.6 &#181;g/&#181;L), anti-mouse Ly6C APC (2 &#181;g/&#181;L), and anti-mouse Ly6G Pe/Cy7 (2 &#181;g/&#181;L). Cover the tubes with aluminum foil to protect the fluorescent antibodies from light and incubate the tubes in the refrigerator at 4 &#176;C for 45 min. 
	NOTE: For single-stain compensations, prepare a negative control tube (no stain) and tubes with cells labeled singly with each of the fluorochromes. To help set up the gate, isotype controls can be used especially for Ly6C (IgG2a) and Ly6G (IgG2b).</li>
	<li>Wash the cells twice with RPMI medium and spin them at 400 x g for 5 min at 4 &#176;C. Count the cells using trypan blue and a hemocytometer and dilute them in 1 mL of RPMI medium per 1 x 106 cells. Before sorting, transfer the samples through a 5 mL, 70 &#181;m cell strainer tube cap. Add DAPI (1 &#181;g/mL) as a cell viability marker and proceed to sort.</li>
	<li>Because macrophages are sensitive and fragile cells, set the sort conditions to 20 psi and use a 100 &#181;m nozzle size to isolate macrophages using a 4-way purity mode.</li>
	<li>Prepare collection tubes with 1 mL of RPMI medium in a 5 mL polypropylene tube.</li>
	<li>Using the sorter software open new experiment and select blank experiment with a blank panel to define settings.</li>
	<li>Set up a dot plot that displays the forward (FSC) versus (vs.) side scatter (SSC) and gate on all leukocytes, excluding debris and clumps with the lowest forward and side scatter. From this parent gate, create a new dot plot that displays SSC vs. DAPI and gate on DAPI cells.
	<ol>
		<li>On this newly-gated population, create a dot plot that displays CD11b vs. CD45 and gate on double-positive (CD11b+ CD45+) macrophages and neutrophils.</li>
		<li>From here, create a final dot plot displaying Ly6C vs. Ly6G and gate the desirable populations: Ly6Chi Ly6G-, Ly6CloLy6G-, and Ly6CintLy6G+ (Figure 2).</li>
	</ol>
	</li>
	<li>After sorting, check for purity and cell viability (&#62;90%) and spin the collection tubes at 400 x g for 5 min at 4 &#176;C. Count the cells using trypan blue and a hemocytometer and resuspend them at the desired concentration (e.g., 1 x 106 macrophages/mL) in complete RPMI medium.</li>
	<li>Plate 50 x 103 macrophages per well in a 96-well round-bottom plate with a final volume of 100 &#181;L of complete RPMI medium. Leave the cells undisturbed for at least 24 h at 37 &#176;C and 5% CO2.</li>
</ol>
4. Isolation of T Cells Using Fluorescence-activated Cell Sorting
NOTE: C57BL/6-Foxp3tm1Flv/Jn is an X-linked targeted knock-in mouse strain that co-marks cells expressing the Foxp3 (forkhead box P3) gene with monomeric red fluorescent protein (mRFP).
<ol>
	<li>Anesthetize and sacrifice C57BL/6 and C57BL/6-Foxp3tm1Flv/J (H-2b) mice as previously described in step 2.1. Isolate the spleen and lymph nodes (LN: inguinal, lumbar, axillar, brachial, and cervical) and rapidly place them in ice-cold RPMI medium.</li>
	<li>In order to isolate the LN, place the mouse in a supine position, make a midline skin incision from bottom to top, carefully cutting the peritoneum and gently spreading the skin apart. Once all inguinal, lumbar, axillary, brachial, and cervical LN are collected (Figure 1C, colored in red), cut the peritoneum and isolate the spleen at the upper left side of the gut.</li>
	<li>Disaggregate the spleen and LN using a 100 &#181;m filter placed on top of a 50 mL tube. Using the plunger of a 1-mL syringe, gently press the tissue. Rinse the filter with RPMI medium as many times as necessary until it is clean. 
	NOTE: LN and spleen from each mouse can be pooled together in the same tube.</li>
	<li>Spin down at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the pellet in 2 mL of ACK lysis buffer. Mix well and incubate for 5 min at 4 &#176;C.</li>
	<li>Add 2 mL of RPMI medium to neutralize the lysis buffer. Spin down at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 200 &#181;L of RPMI medium and transfer it to a 5 mL polystyrene round-bottom tube.</li>
	<li>Stain for anti-mouse CD4 APC (0.6 &#181;g/&#181;L) and/or anti-mouse CD8 PeCy7 (2 &#181;g/&#181;L). Cover the tubes with aluminum foil to protect the fluorescent antibodies from light, and incubate the tubes in the refrigerator at 4 &#176;C for 45 min. 
	NOTE: For single-stain compensations, prepare a negative control tube (no stain) and tubes with cells labeled singly with each of the fluorochromes.</li>
	<li>Wash the cells twice with RPMI medium and spin them at 400 x g for 5 min at 4 &#176;C. Count the cells using trypan blue and a hemocytometer. 
	NOTE: CFSE dye is provided as carboxyfluorescein diacetate succinimidyl ester powder usually at 50 &#181;g. Add 18 &#181;L DMSO to a vial of CFSE for a final stock solution of 5 mM and store at -20 &#176;C for several months.</li>
	<li>For CFSE labeling, resuspend CD8 stained cells at a concentration of up to 106 cells per mL in PBS at a final working concentration of 5 &#181;M CFSE from the stock solution. Incubate them in a bath at 20 &#176;C for 5 min as previously described6. 
	NOTE: (CRITICAL STEP) For cells at a low concentration (&#8804;106 per mL), it is essential that the cells be labeled in the presence of added protein to buffer the toxic effects of CFSE. Therefore, PBS can contain 5% FBS. Note that if medium is used, free amino acids may lower the labeling efficiency by competing for CFSE.</li>
	<li>Neutralize the CFSE with 2 mL of RPMI medium. Spin at 400 x g for 5 min at 4 &#176;C. Add 1 mL of RPMI medium per 1 x 106 CD8 T cells.</li>
	<li>Before sorting, transfer the sample through a 70 &#181;m cell strainer on a 5 mL tube cap. Add 50 &#181;L of 1x DAPI (1 &#181;g/mL) as a cell viability marker and proceed to sorting. 
	NOTE: 1x means 1:1 ratio of the reagent with respect other constituent.</li>
	<li>Set the sort conditions at 20 psi and 100 &#181;m nozzle size to isolate double-positive CD8+CFSE+ T cells (Figure 3A).</li>
	<li>Prepare collection tubes with 1 mL of RPMI medium in a 5 mL polypropylene tube and proceed to sort.</li>
	<li>Using the sorter software open new experiment and select blank experiment with a blank panel to define settings.</li>
	<li>For CD4+ T cell isolation, set up a dot plot that displays the FSC vs. SSC and gate on lymphocytes, excluding debris as well as large granular cells, such as dendritic cells.
	<ol>
		<li>From this parent gate, create a new dot plot that displays SSC vs. DAPI and gate on DAPI cells.</li>
		<li>On this newly-gated population, create a dot plot that displays SSC vs. CD4 to isolate all CD4+ cells (Figure 3B). Alternatively, an anti-CD3 mAb can be used to ensure isolation of pure T cell populations. 
		NOTE: A small fraction of all CD4+ T cells (5-10%) should be Foxp3+mRFP+.</li>
	</ol>
	</li>
	<li>After sorting, check for purity and cell viability (&#62;90%) and spin the collection tubes at 400 x g for 5 min at 4 &#176;C. Count the cells using trypan blue and a hemocytometer. Resuspend using 1 mL of complete RPMI medium per 2x106 T cells.</li>
	<li>Set up suppression assay by culturing T cells with previously-sorted macrophages. Plate 10 x 104 CD4+ T and 10 x 104 CD8+CFSE+ T cells in the same well in a final volume of 100 &#181;L of complete RPMI medium. Incubate at 37 &#176;C and 5% CO2 for 96 h. 
	NOTE: Macrophages and T cells were used in a 1:4 ratio.</li>
	<li>Stimulate T cell division by washing T cell-activator CD3/CD28 magnetic beads in 2 mL of PBS before using them in order to clear the azide-containing buffer. Add 5 &#181;L of mouse T cell-activator CD3/CD28 beads per well. 
	NOTE: Magnetics beads are similar in size to antigen-presenting cells and are coupled to anti-CD3 and anti-CD28 mAb, offering a simple method for the activation and expansion of T cells.</li>
</ol>

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	<li>Jose, M. D., Ikezumi, Y., van Rooijen, N., Atkins, R. C., Chadban, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+act+as+effectors+of+tissue+damage+in+acute+renal+allograft+rejection.">Macrophages act as effectors of tissue damage in acute renal allograft rejection.</a> Transplantation. 76 (7), 1015-1022 (2003).</li><li>Oberbarnscheidt, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-self+recognition+by+monocytes+initiates+allograft+rejection.">Non-self recognition by monocytes initiates allograft rejection.</a> J Clin Invest. 124 (8), 3579-3589 (2014).</li><li>Garcia, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytic+suppressive+cells+mediate+cardiovascular+transplantation+tolerance+in+mice.">Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice.</a> J Clin Invest. 120 (7), 2486-2496 (2010).</li><li>Conde, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-SIGN(+)+Macrophages+Control+the+Induction+of+Transplantation+Tolerance.">DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.</a> Immunity. 42 (6), 1143-1158 (2015).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nat Immunol. 17 (1), 34-40 (2016).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> J Vis Exp. (88), e51627 (2014).</li><li>Tario, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+staining+and+proliferation+modeling+methods+for+cell+division+monitoring+using+cell+tracking+dyes.">Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.</a> J Vis Exp. (70), e4287 (2012).</li><li>Poujol, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+evaluation+of+lymphocyte+transformation+test+based+on+5-ethynyl-2'deoxyuridine+incorporation+as+a+clinical+alternative+to+tritiated+thymidine+uptake+measurement.">Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.</a> J Immunol Methods. 415, 71-79 (2014).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cell Immunol. 256 (1-2), 79-85 (2009).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp. (6), e238 (2007).</li><li>Ochando, J., Conde, P., Bronte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Derived+Suppressor+Cells+in+Transplantation.">Monocyte-Derived Suppressor Cells in Transplantation.</a> Curr Transplant Rep. 2 (2), 176-183 (2015).</li><li>Gallina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+induce+a+subset+of+inflammatory+monocytes+with+immunosuppressive+activity+on+CD8++T+cells.">Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.</a> J Clin Invest. 116 (10), 2777-2790 (2006).</li><li>Huang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gr-1+CD115++immature+myeloid+suppressor+cells+mediate+the+development+of+tumor-induced+T+regulatory+cells+and+T-cell+anergy+in+tumor-bearing+host.">Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.</a> Cancer Res. 66 (2), 1123-1131 (2006).</li><li>Luan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytic+myeloid-derived+suppressor+cells+accumulate+in+renal+transplant+patients+and+mediate+CD4(+)+Foxp3(+)+Treg+expansion.">Monocytic myeloid-derived suppressor cells accumulate in renal transplant patients and mediate CD4(+) Foxp3(+) Treg expansion.</a> Am J Transplant. 13 (12), 3123-3131 (2013).</li><li>Tario, J. 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The authors have nothing to disclose.

This protocol describes the methods we used to immunocharacterize graft-infiltrating myeloid cell subsets in an experimental murine model of heart transplantation, which is also applicable to other tissues in different murine experimental models. Low-pressure cell sorting at 20 psi was the preferred method to isolate a good yield of pure cell subsets. Maintaining the purity of each myeloid subset is critical to establish conclusive results of the suppressive capacity between the different myeloid populations. However, other methods can be used for the isolation of various leukocyte populations, such as commercial enrichment kits. Regardless of the cell population of interest, obtaining viable single-cell suspensions from the transplanted tissue is required for optimal surface staining and flow cytometry results. Incorrect tissue manipulation may cause cell loss and therefore a low yield of myeloid subsets. To increase the yield, make sure to process the sample using cold buffers and to maintain the cells in containers with low cell adherence (polypropylene tubes). In this protocol, we used Collagenase A from C. histolyticum, which is widely used for the disaggregation of many types of tissues (e.g., lung, heart, muscle, bone, adipose, liver, kidney, cartilage, mammary gland, placenta, blood vessel, brain, and tumor). To prevent the loss of cells while sorting, optimizing the antibodies and generating an efficient gating strategy is highly recommended. In this protocol, we characterized murine macrophages using CD11b-Percp/Cy5.5, CD45-APC/eFluor780, Ly6C/APC, and Ly6G Pe/Cy7 fluorescent-conjugated antibodies and flow cytometry. Since the epitopes Ly6C/G do not exist in human, the use of CD14/CD15/CD16 is required for sorting human myeloid cells15.
In multicolor flow cytometry analysis, possible spectral overlaps of the chosen fluorochromes may occur. Therefore, as noted before, single-stain compensations are very helpful to avoid overlapping issues. In addition, antibody titration is highly recommended in order to reduce the background fluorescence and to get optimal results. Another important consideration to reduce non-specific binding is the use of viability dyes. In this protocol, DAPI is used as viability dye. DAPI (4&#39;, 6-diamidino-2-phenylindole) is a blue fluorescent stain that binds strongly to AT-rich regions in DNA. The excitation maximum for DAPI bound to DNA is 358 nm, and the emission maximum is 461 nm. When used according to protocol, DAPI stains nuclei specifically, with little or no cytoplasmic labeling, excluding dead cells in the flow analysis. However, depending on the panel of antibodies chosen, other viability dyes, such as 7-Aminoactinomycin D (7-AAD) and propidium iodide (PI), may be used. Accurate dead-cell exclusion and live-cell identification is a prerequisite to successfully monitor in vitro T cells, as it is important to have procedures that can follow lymphocyte proliferation with minimal disruption to cell viability and function.
As mentioned above, there are critical steps for cell labeling and proliferation analyses with cell tracking dyes such as CFSE. As an example described in the literature, careful attention must be paid to the exclusion of dead/dying cells in order to obtain uniform distributions and distinguishable daughter peaks when using CFSE16. There are many commercially available cell tracking dyes to choose from, depending on the staining panels. As an alternative to cell tracking dyes, thymidine titriated assay are performed to evaluate the proliferation of lymphocytes and other cells in cancer studies13,14. Thymidine incorporation protocols assess the replication of DNA during cell division by the measure of radiolabeled 3H- or 14C- thymidine. Although this method is quite sensitive and well established, the major disadvantages for thymidine titrated technique is that it involves radioactivity and does not provide information at the single cell level. In the other hand, CFSE flow cytometry analysis provides clear information about responding lymphocyte subsets and has less inter and intralaboratory variability.

This protocol describes in vitro techniques to study the function of tissue-infiltrating macrophages isolated from cardiac allografts, according to their ability to modulate T-cell responses. Widely described in the literature, fluorescent cell-tracking dyes in combination with flow cytometry, are powerful tools to study the suppressive function of specific cell types in vitro and in vivo. Our protocol follows the carboxyfluorescein succinimidyl ester (CFSE) method for monitoring lymphocyte proliferation in vitro.
When a CFSE-labeled cell divides, its progeny acquires half the number of carboxyfluorescein-tagged molecules6. The corresponding decrease in cell fluorescence by flow cytometry can be used to assess cell division, monitoring the capacity of suppressive macrophages to modulate T-cell immune responses. Since CFSE is a fluorescein-based dye, it is compatible with a broad range of other fluorochromes, making it applicable to multi-color flow cytometry. The appropriate choice of fluorochromes for phenotyping is also important to avoid excessive spectral overlap and the inability to recognize antibody-positive cells, particularly with visible protein dyes such as CFSE7.
There are many advantages of using fluorescent dyes over alternative techniques that measure cell proliferation, such as the thymidine incorporation assay, which uses radiolabeled thymidine (TdR)8. This assay utilizes tritiated thymidine (3H-TdR) that is incorporated into new strands of chromosomal DNA during mitotic cell division. A safety concern associated with this assay is the use of radioisotopes, since a scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells in order to determine the extent of cell division. Methodologically, the tritiated thymidine assay is not flexible enough to fit important clinical laboratory constraints such as low number of cells and delayed analysis after staining. On the contrary, CFSE staining has been shown to prevent cell proliferation and to interfere with critical activation markers, such as CD69, HLA-DR and CD259. Therefore, understanding advantages and limitations of each methodology, particularly in multicolor studies where multiple dyes are used to track different cell types, is critical for obtaining accurate and reproducible results.

<table><tbody><tr><td>RPMI 1640 Media</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>10% FBS</td><td>Life Technologies</td><td>26140-079</td><td></td></tr><tr><td>1,000x 2-mercaptoethanol</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>100x Pen/Strep</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>100x L-glutamine</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>100x Non Esential amino acids</td><td>Corning</td><td>25025039</td><td></td></tr><tr><td>1 M HEPES buffer&amp;nbsp;</td><td>Corning</td><td>25060CL</td><td></td></tr><tr><td>100 mM Sodium Pyruvate</td><td>ThermoScientific</td><td>SH30239.01</td><td></td></tr><tr><td>Penicilin/Streptavidyn</td><td>ThermoScientific</td><td>15140122</td><td></td></tr><tr><td>Collagenese A</td><td>Roche</td><td>70381322</td><td></td></tr><tr><td>DPBS (w/o calcium&amp;amp;magnesium)</td><td>Corning</td><td>21031CV</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22363548</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Life Technologies</td><td>A10492-01</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>32670-5mg-F</td><td></td></tr><tr><td>CFSE-FITC</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>Dynabeads Mouse T cell activator CD3/CD28</td><td>Life Technologies</td><td>11452D</td><td></td></tr><tr><td>96 well&amp;nbsp; U-bottom plate</td><td>Corning</td><td>353077</td><td></td></tr><tr><td>Antibodies:</td><td></td><td></td><td></td></tr><tr><td>anti-Ly6C-APC</td><td>eBioscience</td><td>175932-80</td><td></td></tr><tr><td>anti-Ly6G-Pe/Cy7</td><td>Biolegend</td><td>127617</td><td></td></tr><tr><td>anti-CD11b-Percp/Cy5.5</td><td>eBioscience</td><td>45011282</td><td></td></tr><tr><td>anti-CD45-APC/Cy7</td><td>eBioscience</td><td>47045182</td><td></td></tr><tr><td>anti-CD4-APC</td><td>eBioscience</td><td>17004181</td><td></td></tr><tr><td>anti-CD8-P/ Cy7</td><td>eBioscience</td><td>25008181</td><td></td></tr><tr><td>LSRII Flow Cytometer</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>FACSDiva Software</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>C57BL/6-Foxp3tm1Flv/J&amp;nbsp;</td><td>The Jackson Laboratory</td><td>008374</td><td></td></tr><tr><td>C57BL/6</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Balb/c</td><td>The Jackson Laboratory</td><td>000651</td><td></td></tr></tbody></table>

functional characterization of regulatory macrophages that inhibit graft-reactive immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

The representative results show the gating strategy described in the above protocol. Results also display the analysis of the T-cell proliferation activity after the co-culture with graft-infiltrating macrophages. The in vitro suppressive capacity of macrophage subsets was analyzed in Figure 4. The results indicate that the Ly6CloLy6G- macrophages obtained from tolerized recipients are suppressive. The results also indicate that Ly6CintLy6G+ cells display a modest suppressive capacity. Only Ly6CloLy6G- cells promoted the expansion of CD4+Foxp3+ Treg in vitro. Together, the data support the conclusion that graft-infiltrating CD11b+Ly6CloLy6G- macrophages possess many of the properties reported to be associated with monocyte-derived suppressor cells12, including their ability to inhibit CD8 T-cell proliferation13 and to promote CD4+Foxp3+ Treg expansion14.
<img alt="Figure 1" src="/files/ftp_upload/54242/54242fig1.jpg" /> 
Figure 1: Animal model. (A) Balb/c hearts (H2-d) were transplanted into fully allogeneic C57BL/6 (H2-b) as as previously described10. Anastomosis, of the recipient&#39;s cava vein and abdominal aorta with the donor&#39;s pulmonary artery and ascending aorta respectively, is shown. Recipient mice were treated with 250 &#181;g of anti-CD40L mAb (clone MR1) for tolerance induction on days 0, 2, and 4, as we recently reported4. Graft function was monitored every other day by abdominal palpation. Rejection was defined as complete cessation of a palpable heartbeat and was confirmed by direct visualization at laparotomy. (B) Representative Image and (C) illustration of anatomical location of LN and spleen (colored in red) and the allograft (colored in purple) <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54242/54242fig2.jpg" /> 
Figure 2: Macrophage sorting strategy. Starting from the upper left, leukocytes are first gated by size, and then singlet cells are discriminated from debris and clumps. From singlets, dead cells are excluded gating on the DAPI negative fraction. From live cells, CD45+CD11b+ is used to identify myeloid cells. Three myeloid cells populations are further identified based on Ly6C and Ly6G expression. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54242/54242fig3.jpg" /> 
Figure 3: Lymphocyte sorting strategy. Representative flow cytometry results for the lymphocyte sorting strategy. From the upper left, gate on the lymphocyte population according to forward and side scatter. Exclude debris and clumps. From singlets, dead cells are excluded by gating on the DAPI negative fraction. From live cells, (A) CD8+ CFSE+ double-positive cells and (B) all CD4+ T cells, which contain a fraction of Foxp3+ positive cells, are gated. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54242/54242fig4.jpg" /> 
Figure 4: Suppression assay. (A) In vitro suppressive capacity of each myeloid subset. CD8+ T-cell proliferation was monitored by CFSE dilution after 96 h of co-culture with myeloid subsets. (B) In vitro Treg expansion of each myeloid subset. Flow cytometry analysis of Foxp3 expression on CD4+ T cells after 96 h of co-culture with myeloid subsets. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54242/54242fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

functional-characterization-regulatory-macrophages-that-inhibit-graft

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.2K Views.  Icahn School of Medicine at Mount Sinai. Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Video: Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Medicine

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Summary

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Chapters in this video

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Depletion and Reconstitution of Macrophages in Mice

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging