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

Summary

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

Abstract

Tissue-resident memory T cell (T_RM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate T_RMs. 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 T_RMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in T_RM-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.

Introduction

Tissue-resident memory (T_RM) T cells represent one of the most abundant T cell populations in adult human and infected mice. T_RM cells provide the first line of immune defense and are critically involved in various physiological and pathological processes^1,2,3,4,5. Comparing with circulating T cells, T_RM cells carry distinct surface markers with unique transcription programs^6,7,8. E....

Protocol

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

1. 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 transferring into female mice will result in immune-mediated rejection of circulating donor T cells in around 12 days¹⁴.
2. P....

Results

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.......

Discussion

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 T_RM biology across multiple tissues. Here, we described a protocol to analyze CD8⁺ T cells in the kidney. This protoco.......

Materials

List of materials used in this article

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