Name: reliable and high efficiency extraction of kidney immune cells
Uploaded: 2016-08-19T13:00:00
Description: Watch this Scientific Journal Video about Reliable and High Efficiency Extraction of Kidney Immune Cells at JoVE.com

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

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&#160;pneumothorax.
1. Perfusion of the Kidneys&#160;
Note: Perfusion of organs such as heart, liver and kidney remo...

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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 ...

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. These immune cells secrete both pro-inflammatory and anti-inflammatory cytokines and chemokines to orchestrate the process of repair 5,12. Currently, the ability to use multiple fluorophores simultaneously to label cell populations in a single cell suspension has increased with the advent of modern flow cytometry machines with four to five lasers. This has substantially added to the capability to discriminate the cell populations based on their functional status 3,7. For example, to accurately label a macrophage as F4/80lowCD11bhighLy6bhighCD206low, at least 3 more fluorophores would be needed in the same sample volume to gate for live cells, CD45+ (leukocytes) and Ly6G- (neutrophils) and this is very much possible with the newer Flow Cytometers 3. However, the downstream assays for cytokine secretion, cell proliferation, cytotoxicity, macrophage activation and the quantification of numbers of various subsets of lymphocytes and monocytes not only needs good quality (live cells, singlets) but adequate numbers of cells.
The immune system in the kidney is made up of both adaptive and innate components and multiple cell types 1,7,13. For example, in mice the two kidneys together are reported to contain 2-17% (28,000-266,000) CD45+ cells of the total kidney immune cells isolated (1.4 x 106 cells) and about 5-15% (1,400-4,200) of these are CD4+ cells 1,5,12. A small percentage (5-15%, 70-630) of these CD4+ cells are FoxP3+ cells (Figure 1)1. Due to these step wise reductions in percentages of cells, sometimes the cell population of interest (in this case CD45+CD4+FoxP3+ cells) is represented by a mere ~100 cells. The small number of CD45+CD4+FoxP3+ cells makes it imperative that a large number of total cells are isolated and the cells are of good quality for downstream studies such as cytokine secretion assays. Moreover, it may be necessary to combine kidneys from 2-3 mice since the subpopulations are not represented in high enough numbers to perform quantifiable assays. Hence, reliable and efficient isolation of kidney mononuclear cell populations is desirable in order to study the immunological spectrum associated with kidney diseases.
Traditionally, for isolation of kidney mononuclear cells, investigators have used a variety of enzymatic digestions such as collagenase 1A or II including DNase 1 1,5,12. It is well known that collagenases have enzymatic activity that varies with lot numbers and by company of manufacture, necessitating titration for the optimum concentration and duration of incubation 4,14,15. In addition, digestion with collagenase adds time for mincing the kidney into small pieces, necessitates incubation of the kidney pieces in a heated (37 &#176;C) bath and additional time for incubation in EDTA for stopping the reaction. In addition, less sterility may be achieved for some downstream procedures needing cell culture. More importantly, depending on the investigator and all the variables involved, it leads to variability in the data and interpretation across laboratories. Recently, with the development of mechanical tissue disruptors/homogenizers 16, the collagenase digestion step is completely avoided and replaced by a simple mechanical disruption of the kidneys 2. Herein, we demonstrate a simple yet efficient method for the isolation of kidney immune cells for everyday immune cell extractions. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain 16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Stomacher 80 Biomaster lab system</td>
			<td>Seward</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher 80 Classic bags</td>
			<td>Seward</td>
			<td>BA6040/STR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XFR Centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td>63514-11</td>
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		</tr>
		<tr>
			<td>Analytical flow cytometer</td>
			<td>BD LSR-X20 Fortessa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>Sigma</td>
			<td>P1644</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline 1X (DPBS)</td>
			<td>Gibco, Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes, no cap</td>
			<td>Becton Dickinson</td>
			<td>352002</td>
			<td></td>
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		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences&#160;</td>
			<td>FVS510,&#160; 564406</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 (Clone: 93)</td>
			<td>EBioscience</td>
			<td>14-0161</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 (clone: 30-F11)&#160; BV421&#160;</td>
			<td>BD Pharmingen</td>
			<td>103133/4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Foxp3 (Clone: FJK-16s) APC</td>
			<td>EBioscience</td>
			<td>17-5773</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD127 (Clone: A7R34) PE/Cy7</td>
			<td>Biolegend</td>
			<td>135013/4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD44 (Clone: IM7) PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>103031/2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD4 (Clone: RM4-5) APC-Cy7</td>
			<td>Biolegend</td>
			<td>100413/4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD8 (Clone: 53-6.7) BV785</td>
			<td>Biolegend</td>
			<td>100749/50</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G (Clone: 1A8) FITC</td>
			<td>Biolegend</td>
			<td>127605/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD11b (Clone: M1/70) PerCP-Cy5.5</td>
			<td>Biolegend</td>
			<td>101227/8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-F4/80 (Clone: BM8) APC</td>
			<td>Biolegend</td>
			<td>123115/6</td>
			<td></td>
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			<td>Anti-CD11c (Clone: N418) BV785</td>
			<td>Biolegend</td>
			<td>117335/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD301 (Clone: LOM-14) PE-Cy7</td>
			<td>Biolegend</td>
			<td>145705/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD26 (Clone: H194-112) PE</td>
			<td>Biolegend</td>
			<td>137803/4</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Tubes 50 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand Tubes 15 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette fine tip</td>
			<td>Samco Scientific</td>
			<td>232</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>Flow Cytometery Staining Buffer Solution</td>
			<td>EBioscience</td>
			<td>00-4222-26</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr>
			<td>1X RBC Lysis Buffer</td>
			<td>EBioscience</td>
			<td>00-4333-57</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
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reliable and high efficiency extraction of kidney immune cells

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.

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...

Watch this Scientific Journal Video about Reliable and High Efficiency Extraction of Kidney Immune Cells at JoVE.com

Reliable and High Efficiency Extraction of Kidney Immune Cells

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 ...

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