Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Summary

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

Abstract

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases^1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension⁶. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention^1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

Introduction

Recent evidence demonstrates that the adaptive immune system, particularly T lymphocytes, play a critical role in the development of several cardiovascular diseases^1,2,3,4,5. For example, in the model of angiotensin II induced hypertension, an accumulation of T cells in the vessels and kidneys of mice has been described⁶. The vascular accumulation is predominantly in the adventitia and the perivascular fat. In the kidney, T cells accumulate in both the medulla and renal cortex. Depending on which subset is involved, these T cells give rise to different cytokines that can affect vascular and renal function and lead to the development of pathology ....

Protocol

Vanderbilt University's Institutional Animal Care and Use Committee has approved the procedures described herein. Mice are housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press. Revised 2010).

1. Isolation of the Aortic Draining Lymph Nodes, Kidney and Aorta from Mice

1. Euthanize the mice by CO₂ inhalation. Spray the chest with 70% ethanol and carefully open the skin and the chest wall with scissors to expose the heart.
2. To perfuse the vasculature, perform a small incision in the right atrium and steadily inject at least 10 ml of cold PBS (approximately 1 ml/sec) ....

Results

The protocol described permits the identification of surface and intracellular markers in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. Representative results are presented below.

Figure 1 demonstrates the gating strategy used to identify the T cell population in a single cell suspension prepared from the aorta of a WT mouse infused .......

Discussion

The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue⁹. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase D	ROCHE	11088882001
Collagenase A	ROCHE	10103586001
Collagenase B	ROCHE	11088815001
Dnase	ROCHE	10104159001
1x Red blood cell lysis buffer	eBioscience	00-4333-57
RPMI Medium 1614 1x	Gibco	11835-030
DPBS without calcium and magnesium	Gibco	14190-144
Percoll	GE Healthcare	17-5445-02	For density gradient centrifugation
GentleMACS™ C tube	Miltenyi Biotec	130-096-334
GentleMACS dissociator device	Miltenyi Biotec	130-093-235	Use the program SPLEEN_04
Cell activation cocktail (with Brefeldin A)	Biolegend	423303
anti-CD16/32	eBioscience	14-0161-81	dilute 1:100
LIVE/DEAD fixable violet dead cell stain kit	Life Technologies	L34955
Transcription factor buffer set	BD Pharmingen	562725
OneComp eBeads	eBioscience	01-1111-42
123 count eBeads	eBioscience	01-1234-42
CD45 AmCyan (clone 30-F11)	BioLegend	103138
CD3 PerCP-Cy5.5 (clone 17A2)	BioLegend	100218
IL-17A FITC (clone TC11-18H10.1)	BioLegend	506910
IL-17F APC (clone 9D3.1C8)	BioLegend	517004
CD4 APC-Cy7 (clone GK1.5)	BD Biosciences	560181
CD8 APC (clone 53-67)	eBioscience	17-0081-82
T-bet PE-Cy7 (clone 4B10)	BioLegend	644823
IFNγ FITC (clone XMG1.2)	BD Biosciences	557724