Name: determination of regulatory t cell subsets in murine thymus, pancreatic draining lymph node and spleen using flow cytometry
Uploaded: 2019-02-27T12:30:00
Description: 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

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 b...

<ol>
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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 ...

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&#228;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>NH4Cl</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&#160;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&#38;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 &#181;m nylon mesh is incorporated into the dual-position snap cap</td>
		</tr>
		<tr>
			<td>Tube 15ml, 120x17mm, PP</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 1.5ml</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+<...

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, ...

This protocol helps in investigating the role of immune cells in particular organs in health and pathology. The main advantage of this technique is that this is a cost-effective technique since it's a manual technique. Demonstrating the procedure will be Zhengkang Luo, a PhD student from my laboratory.To begin, place the thymic glands and spleens from previously euthanized mice into 20 ml scintillation phials, or 15 ml conical tubes, filled with 5 ml of Hank's balanced salt solution. Place the pancreatic draining lymph nodes into 1.5 ml micro-tubes filled with 1 ml of RPMI-1640. Make sure to use the whole thymus and spleen, and all of the PDLNs.Keep the organs on ice throughout the entire procedure. Using a pair of scissors, thoroughly squeeze the thymus and spleen to release the immune cells. Discard the remaining thymic and splenic capsules.Transfer the cell suspension to a 15 ml conical tube. Centrifuge at 433 times g and at 4 degrees celsius for five minutes, and discard the supernatant. To lyse the red blood cells, re-suspend the cell suspension in 5 ml of 0.2 Molar ammonium chloride, and incubate at room temperature for ten minutes.Invert the tubes gently every two minutes. At the end of the incubation, add 5 ml of HBSS to stop the lysis. Centrifuge the tubes at 433 times g and at 4 degrees celsius for five minutes.Discard the supernatant, and re-suspend the cells in approximately 5 ml of HBSS. Then, fill the tubes with HBSS. Repeat this process, from centrifuging the samples to filling the tube with HBSS, one time.Centrifuge the tubes once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in HBSS. Obtain 5 ml round-bottom tubes with cell-strainer caps.Transfer 1 ml of the thymic cell suspension and 500 microlitres of the splenic cell suspension to the tubes by applying the suspension to the cell-strainer caps. First, place a 15 ml conical tube into a rack. Place a sterile 250 micrometer metal mesh over the tube.Rinse the mesh with 1 ml of RPMI. Next, transfer the lymph nodes to the metal mesh and use a pair of tweezers to grind them through the mesh. Apply 1 ml of RPMI on the mesh to flush the cells into the tube.Repeat this process of transferring and grinding the lymph nodes three times for each sample, and then remove the mesh. Centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cells in approximately 5 ml of RPMI.Then, fill the tubes with RPMI. Repeat this process, from centrifuging the samples to filling the tube with RPMI, one time. Centrifuge the tubes again at 433 times g and at four degrees celsius for five minutes.Discard the supernatant and re-suspend the cell pellet in 2 ml of RPMI. Transfer 2 ml of the cell suspension into 5 ml round-bottom tubes with cell-strainer caps. First, centrifuge the cell suspension from the thymus, spleen and PDLN samples at 433 times g and at four degrees celsius for five minutes.Discard the supernatant. Stain the cells with surface antibodies as outlined in the text protocol, and incubate the tubes on ice for 40 minutes. Next, add 200 microliters of FACS buffer to each tube.Centrifuge at 433 times g and at four degrees celsius for five minutes, and discard the supernatant. Repeat this process, adding the buffer, centrifuging, and discarding the supernatant, one time. Re-suspend the cell pellet in 500 microliters of permeabilization fixation buffer to fix and permeabilize the cells.Transfer the tubes to a refrigerator at four degrees celsius overnight. The next day, centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the cell pellet in 500 microliters of permeabilization washing buffer.Centrifuge the cells again at 433 times g and at four degrees celsius for five minutes and discard the supernatant. Stain the cells with intracellular antibodies, as outlined in the text protocol. Incubate the tubes on ice for 1 hour.After this, add 500 microliters of permeabilization washing buffer to each tube. Centrifuge at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in 500 microliters of permeabilization washing buffer.Centrifuge once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cell pellet in 300 microliters of FACS buffer. Then, analyze the cells on a flow cytometer, as outlined in the text protocol.In this study, single cells are isolated from thymic glands, PDLNs and spleens of normal glycemic NOD mice, and are stained with Treg cell markers, CD4, CD25, Foxp3, Helios, and neuropilin-1 for flow cytometric analysis. The results are analyzed and are shown here as representative gating strategies. The proportion of Helios-positive cells among the CD4-positive, CD8-negative, CD25-positive, Foxp3-positive Treg cells is seen to be higher than that of Nrp1-positive cells in all three organs.More than 80%of Treg cells in the thymus are seen to express Helios, which is higher than in the PDLN, and the spleen. The proportion of NrP1-positive cells among Helios-positive Treg cells is seen to be higher in the PDLN than those in either the thymus or spleen. The majority of Nrp1-positive Treg cells also express Helios, and the proportion of Helios-positive cells among Nrp1 positive Treg cells is seen to be higher in the thymus and spleen than in the PDLN.Together, these results indicate that Helios is a better marker to detect T Treg cells than Nrp1. Lymph nodes are small, but some intracellular markers require a large number of cells to give a good signal in flow cytometry. Other methods cannot be performed after this procedure because the cells are already stained and dead.But the animal's four cell markers can be replaced to study other types of immune cells.

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