The present study was supported financially by the Swedish Research Council, EXODIAB, the Swedish Diabetes Foundation, the Swedish Child Diabetes Fund, SEB Diabetesfonden and O.E. och Edla Johanssons vetenskapliga stiftelse. Authors would also like to thanks Per-Ola Carlsson and Stellan Sandler for their supports and discussions.

The local animal ethics committee at Uppsala University approved the animal experiments.
1. Harvesting Organs from Animals
<ol>
	<li>Sacrifice the mice by cervical dislocation.</li>
	<li>Place thymic glands and spleens in 20 mL scintillation vials or 15 mL conical tubes filled with 5 mL of Hanks&#180; balanced salt solution (HBSS). Place PDLNs in 1.5 mL micro tubes filled with 1 mL of RPMI 1640. Use the whole thymus and spleen, and all the PDLNs. 
	NOTE: Organs should be kept on ice throughout the procedure and the investigator should try to be fast while preparing single cell suspensions, especially when handling thymic tissue since thymic immune cells can be quickly degraded at high temperatures.</li>
</ol>
2. Single Cell Isolation from Thymus and Spleen
<ol>
	<li>Thoroughly squeeze the thymus and spleen with a pair of tweezers to release the immune cells. Discard the remaining thymic and splenic capsule.</li>
	<li>Transfer the cell suspension to a 15 mL conical tube and centrifuge at 433 x g for 5 min at 4 &#176;C to form a pellet. Discard the supernatant.</li>
	<li>To lyse the red blood cells, resuspend the cell suspension in 5 mL of 0.2 M NH4Cl and incubate at room temperature for 10 min. Invert the tubes upside down gently every 2 min. Add 5 mL of HBSS at the end of the incubation to stop the lysis.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in approximately 5 mL of HBSS. Fill the tubes with HBSS.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in approximately 5 mL of HBSS. Fill the tubes with HBSS.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in in 5 mL of HBSS for thymus and 10 mL for spleen.</li>
	<li>Transfer 1 mL of the thymic cell suspension and 500 &#181;L of the splenic cell suspension to 5 mL round bottom tubes with cell strainer caps. Apply the cell suspension to the cell strainer caps. 
	NOTE: A 35 &#181;m nylon mesh is incorporated into the cap. Approximately 5-10 million cells were collected in each tube.</li>
</ol>
3. Single Cell Isolation from PDLN
<ol>
	<li>Place a sterile 250 &#181;m metal mesh over a 15 mL conical tube in a rack. Rinse the mesh with 1 mL of RPMI.</li>
	<li>Transfer the lymph nodes to the metal mesh and grind them through the mesh using a pair of tweezers. Apply 1 mL of RPMI on the metal mesh to flush the cells into the tube. Repeat 3 more times and remove the mesh.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 2 mL of RPMI. Transfer 2 mL of the cell suspension to 5 mL round bottom tubes with cell strainer caps. Apply the cell suspension to the cell strainer caps.</li>
</ol>
4. Flow Cytometry
<ol>
	<li>Centrifuge the cell suspensions from thymus, PDLN and spleen at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Stain the cells with the following surface antibodies in 100 &#181;L of flow cytometry staining buffer (FACS buffer): 0.75 &#181;g/100 &#181;L FITC-conjugated CD4 antibody, 0.60 &#181;g/100 &#181;L APC-H7-conjugated CD8 antibody, 0.30 &#181;g/100 &#181;L PE-conjugated CD25 antibody, 5 &#181;L (1: 20) APC-conjugated Nrp1 antibody. Incubate the tubes on ice for 40 min.</li>
	<li>Add 200 &#181;L of FACS buffer to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant. Repeat one more time.</li>
	<li>Fix and permeabilize the cells by resuspending the cell pellet in 500 &#181;L of Permeabilization Fixation Buffer (PFB). 
	NOTE: PFB is prepared by mixing 1 part of Fixation/Permeabilization Concentrate with 3 parts of Fixation/Permeabilization Diluent.</li>
	<li>Keep the tubes in the fridge at 4 &#176;C overnight. 
	NOTE: A 1 h incubation also works.</li>
	<li>Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 500 &#181;L of Permeabilization Washing Buffer (PWB). Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant. 
	NOTE: PWB is prepared by diluting Permeabilization Buffer (10x) to 1:10 in distilled water.</li>
	<li>Stain the cells with the following intracellular antibodies in 100 &#181;L of PWB: 0.30 &#181;g/100 &#181;L PE-Cy7-conjugated Foxp3 antibody, 4 &#181;L (1: 25) PacificBlue-conjugated Helios antibody. Incubate the tubes on ice for 1 h.</li>
	<li>Add 500 &#181;L of PWB to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 500 &#181;L of PWB. Centrifuge the tubes at 433 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 300 &#181;L of FACS buffer.</li>
	<li>Analyze the cells on a flow cytometer.</li>
	<li>Gate the single cells based on Side Scatter-Area, Height and Width, and Forward Scatter-Area, Height and Width. Run one million events for analysis.</li>
	<li>Export the FCS files of experiments and analyze them using a flow cytometry analyzer software.</li>
</ol>

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A., Ramsdell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+Scurfin+in+CD4+CD25++T+regulatory+cells.">An essential role for Scurfin in CD4+CD25+ T regulatory cells.</a> Nature Immunology. 4 (4), 337-342 (2003).</li><li>Hori, S., Nomura, T., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+regulatory+T+cell+development+by+the+transcription+factor+Foxp3.">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science. 299 (5609), 1057-1061 (2003).</li><li>Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+self-tolerance+maintained+by+activated+T+cells+expressing+IL-2+receptor+alpha-chains+(CD25).+Breakdown+of+a+single+mechanism+of+self-tolerance+causes+various+autoimmune+diseases.">Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.</a> The Journal of Immunology. 155 (3), 1151-1164 (1995).</li><li>Vignali, D. A., Collison, L. W., Workman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+regulatory+T+cells+work.">How regulatory T cells work.</a> Nature Review Immunology. 8 (7), 523-532 (2008).</li><li>Thornton, A. M., 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=Expression+of+Helios,+an+Ikaros+transcription+factor+family+member,+differentiates+thymic-derived+from+peripherally+induced+Foxp3++T+regulatory+cells.">Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells.</a> The Journal of Immunology. 184 (7), 3433-3441 (2010).</li><li>Weiss, J. M., 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=Neuropilin+1+is+expressed+on+thymus-derived+natural+regulatory+T+cells,+but+not+mucosa-generated+induced+Foxp3++T+reg+cells.">Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.</a> Journal of Experimental Medicine. 209 (10), 1723-1742 (2012).</li><li>Yadav, M., 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=Neuropilin-1+distinguishes+natural+and+inducible+regulatory+T+cells+among+regulatory+T+cell+subsets+in+vivo.">Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.</a> Journal of Experimental Medicine. 209 (10), S1711-S1719 (2012).</li><li>Singh, K., Hjort, M., Thorvaldson, L., Sandler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+analysis+of+Helios+and+Neuropilin-1+as+a+marker+to+detect+thymic+derived+regulatory+T+cells+in+naive+mice.">Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naive mice.</a> Scientific Reports. 5, 7767 (2015).</li><li>Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+single-cell+suspensions+from+mouse+spleen+with+the+gentleMACS+Dissociator.">Preparation of single-cell suspensions from mouse spleen with the gentleMACS Dissociator.</a> Journal of Visualized Experiments. 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(29), (2009).</li><li>Singh, K., 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=Interleukin-35+administration+counteracts+established+murine+type+1+diabetes--possible+involvement+of+regulatory+T+cells.">Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.</a> Scientific Reports. 5, 12633 (2015).</li><li>Digre, A., 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=Overexpression+of+heparanase+enhances+T+lymphocyte+activities+and+intensifies+the+inflammatory+response+in+a+model+of+murine+rheumatoid+arthritis.">Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis.</a> Scientific Reports. 7, 46229 (2017).</li><li>Li, X., 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=Pro-tumoral+immune+cell+alterations+in+wild+type+and+Shb-deficient+mice+in+response+to+4T1+breast+carcinomas.">Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas.</a> Oncotarget. 9 (27), 18720-18733 (2018).</li></ol>

The authors have nothing to disclose.

In this study, we isolated single cells from thymic glands, PDLNs and spleens of NOD mice, and investigated the expression of Helios and Nrp1 in CD4+CD8-CD25+Foxp3+ Treg cells using flow cytometry. In the present study, NOD mice were used, which is a murine model of type 1 diabetes. In a previous study, we have used the wild type mouse strains CD-1 and C57BL/6 mice to investigate whether Helios or Nrp1 is a better marker for distinguishing tTreg cells from pTreg cells. In that study, we used a similar protocol to determine the subsets of Treg cells in na&#239;ve mice9. We found that Helios is a better marker in this context. Therefore, herein we used normoglycemic NOD mice to confirm our earlier finding in this model and found that the results were consistent with our previous results10. This protocol provides an efficient, yet economical way to prepare single cells and subsequently use them for flow cytometric analysis. There are several published protocols in which single cells are prepared using an automatic dissociator11,12. Instead of using a dissociator, by squeezing the thymic glands and spleens manually and discarding the remaining capsule, we can avoid the presence of fibrous tissue in the cell suspension and make sure the cell suspension is mainly composed of immune cells and red blood cells. We then used NH4Cl to lyse the red blood cells. The duration of lysis can be extended for a few more minutes if the organs are larger than the normal size. However, the lysis duration should not be extended too much, otherwise immune cells will also be lysed. It is important to let the cell suspension go through a cap with cell strainer (or other equivalent cell filters), to prevent clog problems during flow cytometry.
Working in the dark is recommended when working with fluorochrome conjugated antibodies to avoid longer exposure to light. We incubate the cells on ice for extracellular and intracellular staining. The incubation can also be done at room temperature to allow a faster binding of antibodies, but there is a risk of unspecific binding.
Some intracellular markers require large numbers of cells to give good signals in flow cytometry. Given the small size of PDLNs, grinding them on a metal mesh requires more care to allow a maximal release of immune cells. Collecting more PDLNs is also a way to increase the cell number. Another limitation of this protocol is that the cells are fixed and permeabilized for flow cytometric analysis, thus they cannot be used for subsequent functional studies. Magnetic sorting is an option to allow functional studies if only surface markers are of interest.
This protocol has been applied in other mouse models of autoimmunity and cancer13,14. Single cell preparation is a basic but useful technique to determine the characteristics of immune cells in different immune organs. Using this method for single cell preparation might cause necrosis, which may result in decreasing the total number of cells. The spectral overlap of traditional flow cytometry limits the number of markers that can be detected at the same time. Therefore, several panels and large numbers of cells are required to acquire an overall immune profile. In the future, this can be overcome by the development of mass cytometry.

Regulatory T (Treg) cells are critical for the homeostasis of immune system. Treg cells are defined by the expression of surface antigens CD4 and CD25, and the transcription factor forkhead box P3 (Foxp3)1,2,3. Sakaguchi et al. first showed that CD25 is constitutively expressed on T suppressor cells in mice4, which provided a pioneering observation for the identification of human Treg cells. Treg cells play a central role in immune tolerance by exerting a suppressive ability via a variety of mechanisms, including cytolysis through the secretion of granzymes to induce apoptosis, cytokine consumption and inhibition through the expression of cytotoxic T-lymphocyte antigen 45. Additionally, they can secrete inhibitory cytokines such as transforming growth factor beta (TGF-&#946;), interleukin-10 (IL-10) and IL-355. Treg cells can naturally be derived from the thymus, in which case they are designated thymic derived Treg (tTreg) cells, or they can be induced in the peripheral organs in that case are called peripherally induced Treg (pTreg) cells.
Helios, a member of the Ikaros transcription factor family, was reported to be a marker for distinguishing between tTreg cells and pTreg cells6. Later, two other groups reported that Neuropilin 1 (Nrp1), a semaphorin III receptor, could serve as a surface marker for tTreg cells under certain conditions7,8. Nevertheless, Singh et al. have shown that Helios is a better marker in this context in na&#239;ve mice9.
The subsets of Treg cells play a pivotal role in the maintenance of the homeostasis of the immune system and in the protection against autoimmunity and infection. Therefore, it is important to determine the numbers and proportions of these populations in both lymphoid and non-lymphoid tissues. To achieve this, one has to prepare single cell suspensions from these organs. A number of protocols have been described or used in previous studies. Mainly, automatic dissociators have been used in previously published reports10,11. Using automatic dissociators is convenient and time saving, but it is an expensive procedure. Therefore, we have used a manual method to prepare single cell suspensions from murine thymic glands, pancreatic draining lymph nodes (PDLNs) and spleens from normoglycemic non-obese diabetic (NOD) mice, and isolated single cells from these organs. This method has been used in our previous studies and we found that the manual method for preparing single cell suspension is as efficient as automatic dissociator method9,12,13,14. Furthermore, we used flow cytometry to determine the proportions of CD4+CD8-CD25+Foxp3+ Treg cells and the subsets of Treg cells tTreg and pTreg cells. The tTreg and pTreg cells were distinguished using Helios and Nrp1 as markers for tTreg cell. Thus, our results indicate that the manual method for preparing the single cells does work efficiently and the prepared single cell suspensions can be further used for studies using flow cytometry.

<table><tbody><tr><td>NOD mice</td><td></td><td></td><td>In-house breading</td></tr><tr><td>HBSS</td><td>Statens veterin&amp;auml;rmedicinska anstalt</td><td>991750</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R0883-500ML</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>EMD Millipore</td><td>1011450500</td><td></td></tr><tr><td>eBioscience Foxp3 / Transcription Factor Staining Buffer Set</td><td>ThermoFisher</td><td>00-5523-00</td><td>Contains Fixation/Permeabilization Concentrate , Fixation/Permeabilization Diluent and Permeabilization Buffer (10x)</td></tr><tr><td>eBioscience Flow Cytometry Staining Buffer</td><td>ThermoFisher</td><td>00-4222-26</td><td></td></tr><tr><td>CD4 Monoclonal Antibody (RM4-5), FITC, eBioscience</td><td>ThermoFisher</td><td>11-0042-85</td><td></td></tr><tr><td>CD25 Monoclonal Antibody (PC61.5), PE, eBioscience</td><td>ThermoFisher</td><td>12-0251-83</td><td></td></tr><tr><td>BD Pharmingen&amp;nbsp;APC-H7 Rat anti-Mouse CD8a</td><td>BD Biosciences</td><td>560182</td><td></td></tr><tr><td>Mouse Neuropilin-1 APC-conjugated Antibody</td><td>R&amp;amp;D Systems</td><td>FAB5994A</td><td></td></tr><tr><td>FOXP3 Monoclonal Antibody (FJK-16s), PE-Cyanine7, eBioscience</td><td>ThermoFisher</td><td>25-5773-82</td><td></td></tr><tr><td>Pacific Blue anti-mouse/human Helios Antibody</td><td>BioLegend</td><td>137220</td><td></td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>CORNING</td><td>352235</td><td>A 35 &amp;micro;m nylon mesh is incorporated into the dual-position snap cap</td></tr><tr><td>Tube 15 mL, 120 mm x17 mm, PP</td><td>Sarstedt</td><td>62.554.002</td><td></td></tr><tr><td>Micro tube 1.5 mL</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Disposable scintillation vials</td><td>WHEATON</td><td></td><td></td></tr><tr><td>Flow cytometry</td><td>BD</td><td></td><td></td></tr><tr><td>Flow cytometry analysis software</td><td>Inivai Technologies</td><td>Flowlogic</td><td></td></tr></tbody></table>

determination of regulatory t cell subsets in murine thymus, pancreatic draining lymph node and spleen using flow cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

To investigate the expression of Nrp1 and Helios on tTreg and pTreg cells, we prepared single cells from thymic glands, PDLNs and spleens of normoglycemic NOD mice and stained them with the Treg cell markers CD4, CD25, Foxp3, Helios and Nrp1 for flow cytometric analysis. The results were analyzed as shown in the representative gating strategies (Figure 1). We found that the proportion of Helios+ cells among CD4+CD8-CD25+Foxp3+ Treg cells was higher than that of Nrp1+ cells in all three organs (Figure 2A). More than 80% of the Treg cells in the thymus expressed Helios, which was higher than in the PDLN and spleen (Figure 2A). The proportion of Nrp1+ cells among Helios+ Treg cells was higher in the PDLN than those in the thymus and spleen (Figure 2B). The majority of Nrp1+ Treg cells also expressed Helios, and the proportions of Helios+ cells among Nrp1+Treg cells in the thymus and spleen were higher than that in the PDLN (Figure 2C). Together, these results indicate that Helios is a better marker to detect tTreg cells than Nrp1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58848/58848fig1.jpg" /> 
Figure 1: Representative gating strategies for flow cytometry. The live single cells were gated based on FSC and SSC. Then, cells were further gated for CD4 and CD8 expression. CD4+CD8- cells were further gated for the expression of CD25. Next, CD4+CD8-CD25+ cells were gated for Foxp3 expression. CD4+CD8-CD25+Foxp3+ Treg cells were gated for Nrp1 or Helios expression. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58848/58848fig1large.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/58848/58848fig2.jpg" /> 
Figure 2: The expression of Nrp1 and Helios in Treg cells in the thymus, PDLN and spleen. Four 8 week-old non-diabetic NOD mice were sacrificed. Single cells were prepared from the thymic glands, PDLNs and spleens. The cells were then stained with fluorochrome conjugated antibodies for flow cytometric analysis. (A) The percentages of Nrp1+ and Helios+ cells among CD4+CD8-CD25+Foxp3+ Treg cells. (B) The percentages of Nrp1+ cells among Helios+ Treg cells. (C) The percentages of Helios+ cells among Nrp1+ Treg cells. Results are expressed as means &#177; SEM. ***p &#60; 0.001. One-way ANOVA followed by Tukey&#39;s multiple comparisons (A, B and C) and unpaired t-tests (A) were performed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58848/58848fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry at JoVE.com

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

determination-regulatory-t-cell-subsets-murine-thymus-pancreatic

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Immunology and Infection

11.3K Views.  Uppsala University. Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Video: Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Medicine

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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.

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 ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Preparing Mouse Thymic Epithelial Cells for Analysis of Intracellular Molecules

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric AnalysisMechanisms

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

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