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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B., Martin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+kidney+disease+and+immune+modulation.">Diabetic kidney disease and immune modulation.</a> Curr. Opin. Pharmacol. , (2013).</li><li>Kawakami, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+renal+mononuclear+phagocytes+comprise+five+discrete+populations+with+distinct+phenotypes+and+functions.">Resident renal mononuclear phagocytes comprise five discrete populations with distinct phenotypes and functions.</a> J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+lot+selection+and+purification+for+adipose+tissue+digestion.">Collagenase lot selection and purification for adipose tissue digestion.</a> Cell Transplant. 4 (3), 281-289 (1995).</li><li>Yamamoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deterioration+and+variability+of+highly+purified+collagenase+blends+used+in+clinical+islet+isolation.">Deterioration and variability of highly purified collagenase blends used in clinical islet isolation.</a> Transplantation. 84 (8), 997-1002 (2007).</li><li>Zhou, J., Nagarkatti, P., Zhong, Y., Nagarkatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+modulation+by+chondroitin+sulfate+and+its+degraded+disaccharide+product+in+the+development+of+an+experimental+model+of+multiple+sclerosis.">Immune modulation by chondroitin sulfate and its degraded disaccharide product in the development of an experimental model of multiple sclerosis.</a> J. 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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>
			<td>Analytical flow cytometer</td>
			<td>BD LSR-X20 Fortessa</td>
			<td></td>
			<td></td>
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		<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>
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			<td>Polypropylene tubes, no cap</td>
			<td>Becton Dickinson</td>
			<td>352002</td>
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			<td>Fixable Viability Stain</td>
			<td>BD Biosciences&#160;</td>
			<td>FVS510,&#160; 564406</td>
			<td></td>
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			<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>
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			<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>
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			<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 ...

The overall goal of this kidney immune cell isolation protocol is to extract viable single cell suspensions of kidney immune cells in adequate numbers for downstream analysis using multicolor flow cytometry. The main advantage of this technique, compared to collagenase digestion, is the ability to reliably and inexpensively isolate a large number of kidney immune cells for downstream experimental analysis. This method can help answer key questions in the kidney immunobiology field.For example, what subset of kidney immune cells regulate cytokine or chemokine secretion in a specific kidney disease. This method is demonstrated for the isolation of the immune cells from the kidney. However, it can be easily adapted to isolate the cells from other systems like liver or brain.Today, we'll be demonstrating a procedure for collecting kidney immune cells. And the procedure will be demonstrated by Alex Meuth, a research specialist from my lab. After confirming a lack of response to toe pinch, use 70%ethanol to clean the ventral abdomen of an anesthetized mouse.Then, use scissors to open the abdominal cavity, and reflect the abdominal organs to the left side of the animal. Using a one cc syringe equipped with a bent 25 gauge needle, collect the blood from the inferior vena cava into a 1.5 milliliter microcentrifuge tube containing 10 microliters of potassium EDTA. Next, make a small cut in the right atrium, and use a pair of forceps to gently grasp the heart.Then, insert a 21 to 25 gauge needle attached to a 50 cc syringe filled with 20 to 30 milliliters of ice-cold PBS into the left ventricle at its apex, and slowly perfuse the ventricle for one to two minutes. The heart will blanch after the first few milliliters of PBS. Liver blanching indicates that blood is being flushed through the liver via the venous return through the vena cava.When the perfusion is complete, use scissors and forceps to dissect the right kidney from the attached blood vessels, fat, and adrenal glands. Then, gently remove the kidney capsule with forceps, and weigh the kidney as experimentally appropriate. Next, transfer the kidney into a 50 milliliter conical tube containing one milliliter of flow cytometry buffer on ice.Label a five to 80 milliliter capacity homogenization bag with the appropriate sample identification information, and discard the inside strainer. Fill the bag with five milliliters of ice-cold flow cytometry staining buffer, and place the kidney in the bag. Fold the homogenization bag in half, such that one half has the kidney submerged in the buffer, and the other half is folded over it.Place the folded homogenization bag with the kidney facing the paddle close to the bottom edge, taking care that the lower end of the bag does not slide below the lower border of the paddles. Then, visually confirm that the kidneys are adequately homogenized and that no large chunks are visible. If the kidneys are not sufficiently mashed by the instrument, move the paddles closer to the wall of the homogenizer.Then, place one 100 micron cell strainer into an individual 50 milliliter conical tube per sample on ice, and completely filter the homogenates through the strainers. When all of the samples have been dissociated, centrifuge the tubes with the strainers to ensure that all of the tissues are completely filtered, and place the tubes back on ice. Next, add an equal volume of RBC lysis buffer to each sample, and pipette up and down several times to generate uniform cell suspensions.After five minutes, pellet the cells by centrifugation, and carefully discard the supernatant from each tube in one smooth motion, decanting the final drop of liquid that forms while each tube is still upside down. To isolate the kidney immune cells, add one milliliter of 72%density gradient reagent into one round-bottom polypropylene tube for each sample. Next, gently but vigorously triturate one milliliter of 0.25 molar sucrose into each pellet to obtain single cell suspensions, and add the cells to the density gradient solutions.Then, gently layer one milliliter of 72%density gradient reagent under each cell suspension to form distinct heavy layers. Confirm that the kidney cells are uniformly suspended for the 36%percoll gradient in each tube. Then, carefully layer the 72%percoll underneath the 36%percoll to form distinct 36 and 72 layers.Carefully place the tubes in the centrifuge for density gradient separation. Then, use a polypropylene Pasteur pipette to gently but thoroughly transfer all but about the last three millimeters of the upper junk layers into individual empty round-bottom centrifuge tubes. Now, use a fresh Pasteur pipette to gently transfer the buffy coats into new round-bottom centrifuge tubes until three to five millimeters below the buffy coat has been removed to ensure that most of the cells from each sample have been collected.Then, mix one to two milliliters of flow cytometry staining buffer into the buffy coats, and centrifuge the cells. Resuspend the pellets in 500 microliters of flow cytometry staining buffer for counting, and evenly split the samples into a dark, U-bottomed, 96-well plate. Incubate the samples in blocking antibody for 15 minutes at four degrees Celsius.Then, prepare an antibody cocktail for each immune cell type to be tested. For example, lymphocytes, macrophages, and dendritic cells, and incubate the cells in the appropriate antibody cocktail for 30 minutes at four degrees Celsius. After selecting the population of interest by forward and side scatter, exclude the non-viable cells.Two to 18%of the total kidney immune cells typically express CD45. To isolate the effector and cytotoxic T cells, for example, these bone marrow-derived hematopoietic cells can then be gated on CD4 and CD8 respectively. Further characterization of the CD4-positive cells reveals an approximately eight to 14%population of FoxP3-positive regulatory T cells, which can be subdivided into CD44 and CD127 expressing populations.To evaluate the myeloid cell panel, the CD45-positive cells can be gated by their Ly6G expression to first exclude the neutrophils. CD11b and F4/80 can then be used to further subdivide the Ly6C positive cells into the infiltrating macrophage and dendritic cell populations. Once mastered, this technique can be completed in one to two hours.Following this procedure, downstream experiments, such as the isolation of CD11b or CD4-positive cells can be performed to answer questions on cytokine secretion by this specific subset of cells. After watching this video, you should have a good understanding of how to perfuse the kidneys, generate a viable kidney cell suspension, isolate kidney immune cells by gradient density centrifugation, and label the kidney cells for flow cytometric analysis.

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