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Immunology and Infection

Reliable and High Efficiency Extraction of Kidney Immune Cells

Published: August 19th, 2016

DOI:

10.3791/54368

1Division of Nephrology, Department of Medicine, University of Missouri-Columbia and Harry S Truman Memorial Veteran's Hospital, 2Division of Biomedical Sciences, Department of Medicine, University of Missouri-Columbia and Harry S Truman Memorial Veteran's Hospital, 3Division of Endocrinology, Department of Medicine, University of Missouri-Columbia and Harry S Truman Memorial Veteran's Hospital

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes 6,10,11,13. Potential areas of active research encompass the various triggers for immune system activation, various cell types involved, the cytokine/chemokine pattern in a particular disease setting, modulation of all of the aforementioned processes by a particular drug etc. To exemplify, in ischemia-reperfusion injury (a model for acute kidney injury), there is an increase in immune cells or bone-marrow derived hematopoietic cells or CD45+ cells within a few hours, which is sustained through the period of repair or fibrosis (6 weeks later) 5,12

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All protocol steps performed were reviewed and approved by the University of Missouri Animal Care and Use Committee (ACUC). For this protocol, male C57Bl/6 mice aged 15 weeks were utilized although theoretically any rodent at any age can be used for experiments. Since, this is a non-survival surgery, euthanasia is achieved by exsanguination and bilateral pneumothorax.

1. Perfusion of the Kidneys 

Note: Perfusion of organs such as heart, liver and kidney remo.......

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The number of panels that can be run depends on the number of immune cells that can be reliably extracted out of the kidneys. Herein, we demonstrate the ability to run 2 panels, one for T-lymphocytes and one for macrophages/dendritic cells. On the T-lymphocyte panel, we first look at the forward scatter (FSC) and side scatter (SSC) pattern and delineate the population of interest as shown in Figure 1 (top left dot plot). Next, a viability marker, in this case a fixable vi.......

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We have presented here a methodology to obtain immune cells from the kidney in a reliable and efficient manner. The major modification to the widely used collagenase digestion step (mechanical disruption of tissue) saves about 30 min and the isolation of a large number of viable immune cells takes under two hours for 4 kidney samples. Moreover, depending on our research question, we now only use a single kidney (the other kidney can be used for protein analysis by Western blots, immunohistochemistry and mRNA analysis by .......

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This work is supported by a Research Grant from Dialysis Clinics Inc. and from the University of Missouri Research Board Grant.

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Name Company Catalog Number Comments
Stomacher 80 Biomaster lab system Seward
Stomacher 80 Classic bags Seward BA6040/STR
Sorvall Legend XFR Centrifuge Thermo Scientific Or equivalent equipment 
Hemocytometer Electron Microscopy Sciences 63514-11
Analytical flow cytometer BD LSR-X20 Fortessa
Percoll  Sigma P1644
Dulbecco’s phosphate buffered saline 1X (DPBS) Gibco, Life Technologies 14190-250
Polypropylene tubes, no cap Becton Dickinson 352002
Fixable Viability Stain BD Biosciences  FVS510,  564406
Anti-CD16/32 (Clone: 93) EBioscience 14-0161
anti-CD45 (clone: 30-F11)  BV421  BD Pharmingen 103133/4
Anti-Foxp3 (Clone: FJK-16s) APC EBioscience 17-5773
Anti-CD127 (Clone: A7R34) PE/Cy7 Biolegend 135013/4
anti-CD44 (Clone: IM7) PerCP/Cy5.5 Biolegend 103031/2
anti-CD4 (Clone: RM4-5) APC-Cy7 Biolegend 100413/4
anti-CD8 (Clone: 53-6.7) BV785 Biolegend 100749/50
Anti-Ly6G (Clone: 1A8) FITC Biolegend 127605/6
Anti-CD11b (Clone: M1/70) PerCP-Cy5.5 Biolegend 101227/8
Anti-F4/80 (Clone: BM8) APC Biolegend 123115/6
Anti-CD11c (Clone: N418) BV785 Biolegend 117335/6
Anti-CD301 (Clone: LOM-14) PE-Cy7 Biolegend 145705/6
Anti-CD26 (Clone: H194-112) PE Biolegend 137803/4
100 μm filter  Fisher Scientific 22363548
Fisherbrand Tubes 50 ml Fisher Or equivalent equipment 
Fisherbrand Tubes 15 ml Fisher Or equivalent equipment 
Sucrose Fisher chemical S5-3
Transfer pipette fine tip Samco Scientific 232 Or equivalent equipment 
Flow Cytometery Staining Buffer Solution EBioscience 00-4222-26 Or equivalent equipment 
1X RBC Lysis Buffer EBioscience 00-4333-57 Or equivalent equipment 

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