Name: isolation of mouse kidney-resident cd8+ t cells for flow cytometry analysis
Uploaded: 2020-06-27T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis at JoVE.com

This work is supported by NIH grants AI125701 and AI139721, Cancer Research Institute CLIP program and American Cancer Society grant RSG-18-222-01-LIB to N.Z. We thank Karla Gorena and Sebastian Montagnino from Flow Cytometry Facility. Data generated in the Flow Cytometry Shared Resource Facility were supported by the University of Texas Health Science Center at San Antonio (UTHSCSA), NIH/NCI grant P30 CA054174-20 (Clinical and Translational Research Center [CTRC] at UTHSCSA), and UL1 TR001120 (Clinical and Translational Science Award).

All animal experiments performed following this protocol must be approved by the respective institutional Animal Care and Use Committee (IACUC). All procedures described here have been approved by IACUC UT Health San Antonio.
1. Adoptive transfer of P14 T cells into C57BL/6 recipients and LCMV infection
<ol>
	<li>Use P14 mice at 6-12 weeks of age. Ensure that the sex of donor P14 TCR transgenic mouse should match the sex of C57BL/6 recipient mice. Otherwise, cells such as male T cells transf...

<ol>
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As tissue specific immunity is a rapid expanding area of research, accumulating evidence suggest that immune cells, especially lymphocytes population can be identified in almost all organs in adult human or infected or immunized mice. LCMV mouse infection model is a well-established model to study antigen-specific T cell response, effector and memory T cell differentiation including TRM biology across multiple tissues. Here, we described a protocol to analyze CD8+ T cells in the kidney. This protoco...

Tissue-resident memory (TRM) T cells represent one of the most abundant T cell populations in adult human and infected mice. TRM cells provide the first line of immune defense and are critically involved in various physiological and pathological processes1,2,3,4,5. Comparing with circulating T cells, TRM cells carry distinct surface markers with unique transcription programs6,7,8. Expanding our knowledge of TRM biology is the key to understanding T cell responses which is essential for future development of T cell-based vaccines and immunotherapies.
In addition to commonly shared molecular markers of TRMs across all non-lymphoid tissues, accumulating evidence suggest that tissue-specific features are a central component of TRM biology9. Kidney harbors many immune cells including TRM cells after infection and offers a great opportunity to study TRM cell biology in a non-mucosal tissue. Acute LCMV (lymphocytic choriomeningitis virus) infection via intraperitoneal route is a well-established systemic infection model to study antigen-specific T cell responses in mice. The infection is usually resolved in 7-10 days in wild type mice and leave a large number of LCMV-specific memory T cells in a variety of tissues, including the kidney10. P14 TCR (T cell receptor) transgenic mice carry CD8+ T cells recognize one of the immune-dominant epitopes of LCMV presented by MHC-I (class I major histocompatibility complex) molecule H2-Db in C57BL/6 mice. Combining congenically marked P14 T cell adoptive transfer and LCMV infection in mice, CD8+ effector and memory T cells are tracked, including TRM differentiation and homeostasis.
In some barrier tissues, such as intestinal intraepithelial lymphocytes (IEL) compartment and salivary glands, established lymphocyte isolation procedure yields high percentage of TRM cells with minimal blood borne T cell contamination in mouse LCMV model11. However, in non-barrier tissues, such as the kidney, dense vascular network contains a large number of circulating CD8+ T cells. It has been well documented that even successful perfusion cannot efficiently remove all circulating CD8+ T cells. To overcome this technical hurdle, intravascular antibody staining has been established as one of the most commonly used techniques in TRM labs12. In brief, 3-5 minutes before euthanasia, 3 &#181;g/mouse anti-CD8 antibody (to label CD8+ T cells) is delivered intravenously. Intact blood vessel wall prevents rapid diffusion of the antibody within this short period of time (i.e., 3-5 minutes) and only intravascular cells are labeled. Following standard lymphocyte isolation protocol, intravascular versus extravascular cells can be easily distinguished using flow cytometry.
Here, we will describe a standard protocol commonly used in TRM labs to perform intravascular labeling, lymphocyte isolation and flow cytometry analysis of kidney CD8+ T cells using a C57BL/6 mouse that has received CD45.1+ P14 T cells and LCMV infection 30 days before13. Same protocol can be used to study both effector and memory T cells in the kidney.

<table>
	<tbody>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>05-408-130</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Conical Tubes</td>
			<td>Corning</td>
			<td>431470</td>
			<td></td>
		</tr>
		<tr>
			<td>3 ml syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>37C incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin a-CD8a antibody(Clone 53-6.7)</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf Serum</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Collegenase B</td>
			<td>Millipore Sigma</td>
			<td>11088807001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-9AM</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors</td>
			<td>Roboz Surgical</td>
			<td>RS 5692</td>
			<td></td>
		</tr>
		<tr>
			<td>Mojosort Mouse CD8 Na&#239;ve T Cells Isolation Kit</td>
			<td>Biolegend</td>
			<td>480043</td>
			<td></td>
		</tr>
		<tr>
			<td>overhead heat lamp</td>
			<td>Amazon</td>
			<td>B00333PZZG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17089101</td>
			<td></td>
		</tr>
		<tr>
			<td>rocker</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30096.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid Brass Mouse Restrainer</td>
			<td>Braintree Scientific, Inc</td>
			<td>SAI-MBR</td>
			<td></td>
		</tr>
		<tr>
			<td>Swing Bucket Centrifuge with refrigerator</td>
			<td>Thermofisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture 6-well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse kidney-resident cd8+ t cells for flow cytometry analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

The protocol described here is summarized in a flow chart (Figure 1A). At day 30 post LCMV infection, we performed intravascular labeling of CD8+ T cells. 5 minutes later, both kidneys of the animal were dissected, minced and subjected to collagenase digestion. Lymphocytes were further purified from the digested samples via Percoll centrifugation and analyzed by flow cytometry. As shown in Figure 1B, even after density centrifugation-mediated lymphocy...

Watch this Scientific Journal Video about Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis at JoVE.com

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

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