Name: reliable and high efficiency extraction of kidney immune cells
Uploaded: 2016-08-19T13:00:00.000+00:00
Duration: 9 min 15 s
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 removes the blood which may interfere with the interpretation of data. Hence, if possible we always perfuse the organs.
<ol>
	<li>Place the mouse in an isoflurane (3 ml/min) induction chamber till it is immobile. Next, remove the mouse out of the chamber, lay it dorsally on the dissecting table and place the isoflurane cone on the mouse nose and push isoflurane at the rate of 1.5 ml/min through the regulator.</li>
	<li>Check the toe pinch response with tweezers to assess if the animal has achieved a surgical plane of anesthesia. Clean the ventral abdomen with saline and cut open the abdomen with scissors and push the abdominal organs (out of the surgical field) to the left side of the mouse.</li>
	<li>Collect blood from the inferior vena cava with a 25 gauge needle-fixed to a 1 cc syringe and add it to a 1.5 ml microcentrifuge tube containing 10 &#956;l of 0.5M EDTA. 
	Note: Collection of blood is optional. Plasma may be used for biochemical measurements. Leukocytes may be used for flow cytometry assays.</li>
	<li>Using scissors, make a small cut in the right atria to let out the mixture of blood and PBS from the next step.</li>
	<li>Take a 50 cc syringe and fill with ice-cold PBS (20-30 cc). Next, attach a 21-25 G needle to the syringe, puncture the left ventricle at its apex (while holding the heart gently between forceps) and slowly perfuse the left ventricle with phosphate buffered saline pH 7.4 (PBS) over the course of 1-2 min. 
	Note: The heart immediately blanches upon the first few milliliters of perfusion. Observe the liver for blanching, as this indicates that the perfusion is draining blood out of the liver via venous return through the vena cava. In addition, make sure that the lungs are not expanding during the infusion as this indicates puncture of the right ventricle. If the lungs were to expand, withdraw the needle slightly back into the left ventricle and continue the perfusion.&#160;The time taken from the first incision till euthanasia is achieved is less than 2 min</li>
</ol>
2. Dissection of Kidneys&#160;
<ol>
	<li>After perfusion is complete, use scissors and forceps to dissect the right kidney from its attachments (blood vessels, fat and adrenal glands) and excise.</li>
	<li>Gently remove the capsule covering the kidney by cutting and dissecting with a forceps. Weigh the kidney. 
	Note: Weighing the kidney is optional. However, some experiments may result in change in kidney size/weight which may be used to normalize cell yield per gram of tissue.</li>
	<li>After weighing the kidney, place it in 1 ml of RPMI-1640 containing 0.5% FBS or flow cytometry staining buffer solution (50 ml conical screw cap tube) and place the tube on ice till further use. 
	Note: For ease of description, we will describe the rest of the isolation of immune cells in the flow cytometry staining buffer solution. In addition, we will describe isolation of immune cells from a single kidney, although both kidneys or 1+1/3rd of the kidneys can be used depending on the downstream experiments (optional). 1/3rd of the kidney could be frozen for protein/RNA experiments and 1/3rd can be placed in buffers for immunohistochemistry or transmission electron microscopy.</li>
</ol>
3. Homogenization of Kidneys
<ol>
	<li>Label a 5-80 ml capacity homogenization bag with sample identification information, remove the inside strainer (STR) and discard it. Fill the bag with 5 ml of ice cold Flow Cytometry Staining Buffer solution and transfer kidney to the buffer. Repeat this process for a second sample or more samples that need processing.</li>
	<li>Fold the homogenization bag in half, such that one half has the kidney submerged in the Flow Cytometry Staining Buffer solution and the other half is simply folded over it.</li>
	<li>Turn on the tissue homogenizer and set it to fast (setting) rpm. Place the folded homogenization bag with the kidney facing the paddle, close to the bottom edge but make sure the lower end of the bags are not sliding below the lower border of the paddles.
	<ol>
		<li>Process the second sample simultaneously as there are 2 paddles. Select time as 120 sec. Observe the paddles move back and forth to homogenize the kidneys. 
		Note: Meanwhile, this downtime can be used to prepare 2 more kidney (or other tissue) samples while the first two are processing.</li>
	</ol>
	</li>
	<li>After completing with first set of homogenization, transfer the bags to ice and reload the tissue homogenizer with the next set of samples and so on. Visually ensure that the kidneys are adequately homogenized and no large chunks are visible. 
	Note: Generally, 2 min in the tissue homogenizer at fast rpm is adequate for the kidney.</li>
	<li>Place a 100 &#181;m cell strainer into the 50 ml conical tube on ice. Next, pipette the homogenate completely on to the cell strainer. Set a floor centrifuge at 50 x g for 1 min at 4 &#176;C and load the samples into the centrifuge. 
	Note: Centrifugation will ensure that all homogenate (containing cells) has passed through the cell strainer.</li>
	<li>Remove the tubes from the centrifuge and place them back on ice. Discard the cell strainers and keep the homogenate. Add equal volume (~ 6 ml) of 1x RBC lysis buffer to the cell pellet/supernatant and pipette up and down several times to make a uniform cell suspension. Visually, observe some of the red color in the suspension decrease immediately. Leave the tubes on ice for 5 min to achieve maximum RBC lysis. 
	Note: the RBC lysis step can be skipped if the investigator is confident of the kidney perfusion and less cell manipulation is desired. We have observed a significant increase in kidney cell yield by not subjecting the cell suspension to RBC lysis.</li>
	<li>Spin the tubes in the centrifuge that has been set to 500 x g, 4 &#176;C for 5 min to form a cell pellet. 
	Note: During steps 3.6 and 3.7, prepare for colloidal silica (Percoll) based density gradient centrifugation.
	<ol>
		<li>Take 10 ml round bottom polypropylene tubes and label them with corresponding sample numbers and place them in a tube rack. 
		Note: The following describes how to make enough density gradient reagents for 4 kidney samples.</li>
		<li>Take 7 ml of Flow Cytometry Staining Buffer solution and pipette it into a 15 ml conical tube. Add 1 ml of 2 M sucrose to prepare 0.25 M sucrose.</li>
		<li>Next, take 7.2 ml of density gradient reagent and pipette it into another 15 ml conical tube. Add 1.55 ml of Flow Cytometry Staining Buffer solution and 1.25 ml of 2 M sucrose to prepare 72% density gradient reagent and 0.25 M sucrose.</li>
		<li>Pipette 1 ml of 72% density gradient reagent into one round bottom polypropylene tube for each sample.</li>
	</ol>
	</li>
</ol>
4. Gradient Centrifugation
<ol>
	<li>After Step 3.7, take the tubes out and gently decant the supernatant in one smooth motion and also decant the next drop of liquid that forms while the tube is held upside down. 
	Note: This step is important since leaving behind excess liquid can change the density gradient enough to create variation in yields. When the tube is set back upright, ~ 200-300 &#181;l of liquid remains with the cell pellet.</li>
	<li>Pipette 1 ml of 0.25 M sucrose onto the cell pellet and gently but vigorously pipette up and down to bring the cells to a uniform suspension. Next, add the cell suspension to 1 ml 72% density gradient reagent (3.7.4) and once again pipette vigorously to mix the cells to form a 36% density gradient reagent.</li>
	<li>Take 1 ml of the 72% density gradient reagent into a polypropylene Pasteur (transfer) pipette. With even pressure on the bulb (keeps the liquid column in the tip and prevents bubble formation), insert the Pasteur pipette to the bottom of the cell suspension (36% density gradient reagent) gently and unload the 72% density gradient reagent to form a distinct heavier layer.</li>
	<li>Pick the sample tubes gently and place into the centrifuge, which is at room temperature for this step. Spin for 20-30 min at 500 x g.</li>
	<li>Meanwhile, prepare another two sets of polypropylene round bottom centrifuge tubes with the corresponding sample identifiers.</li>
	<li>After Step 4.4 is complete, remove the sample containing polypropylene tubes gently, with minimal shaking and set it down on the tube rack.</li>
	<li>Pick up the first sample tube and its corresponding empty tube. With a polypropylene Pasteur pipette, gently but thoroughly with measured suction, remove the upper &#34;junk layer&#34; or &#34;gooey material&#34; and put it into the empty round bottom centrifuge tube.
	<ol>
		<li>Proceed removing the upper part of the gradient till the &#34;buffy coat&#34; is reached (may appear off-white with a reddish tinge [red blood cells]). Stop suctioning if a height of ~3 mm is reached from the &#34;buffy coat&#34;. 
		Note: Ensure that this stuff is completely gone from the upper part of the gradient before continuing to suction.</li>
	</ol>
	</li>
	<li>Take a fresh Pasteur pipette and a fresh round bottom centrifuge tube and gently remove the buffy coat. Continue to suction 3 - 5 mm below the buffy coat, to ensure that most of the cells are collected and added to the 2nd new round bottom tube with appropriate sample identifier.</li>
	<li>Add 1-2 ml of Flow Cytometry Staining Buffer solution to the 2nd tube and mix thoroughly by pipetting up and down.</li>
	<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C. A 2nd wash step with Flow Cytometry Staining Buffer solution is optional. Resuspend in 500 &#181;l of Flow Cytometry Staining Buffer solution and proceed to count cells using a hemocytometer.</li>
</ol>
5. Optional (Cell Labeling)
<ol>
	<li>Aliqout each sample for the same number of cells. Add the blocking antibody at a dilution of 0.5 &#181;g per 106 cells (anti- CD16/CD32 FcR) and incubate on ice for 15 min.</li>
	<li>Add the desired cocktail of primary antibodies (conjugated to the appropriate fluorophores) that are determined to work well with each other by the investigator, prior to the actual experiments. Incubate on ice for 30 min. Wash the cells with PBS. 
	Note: For the demonstration of kidney immune cell labeling for this manuscript, we used a T-lymphocyte and a macrophage/dendritic cell panel. The T-lymphocyte panel consisted of Fixable Viability Stain FVS510, anti-CD45 (clone: 30-F11) BV421, Anti-Foxp3 (Clone: FJK-16s) APC, Anti-CD127 (Clone: A7R34) PE/Cy7, anti-CD44 (Clone: IM7) PerCP/Cy5.5, anti-CD4 (Clone: RM4-5) APC-Cy7, anti-CD8 (Clone: 53-6.7) BV785. The macrophage panel consisted of Fixable Viability Stain FVS510, anti-CD45 (clone: 30-F11) BV421, Anti-Ly6G (Clone: 1A8) FITC, Anti-CD11b (Clone: M1/70) PerCP-Cy5.5, Anti-F4/80 (Clone: BM8) APC, Anti-CD11c (Clone: N418) BV785.</li>
	<li>Add the Fixable Viability Stain and leave on bench-top for 5 min. Wash the cells with Flow Cytometry Staining Buffer solution to remove excess of the Fixable Viability Stain.</li>
	<li>Add Fixation/Permeabilization reagent and incubate overnight at 4 &#186;C. Wash in permeabilization solution.</li>
	<li>For intracellular stain, block again with anti-CD16/32 FcR for 10min. Add the intracellular stains and incubate for 20-30 min. Wash 2x in permeabilization solution.</li>
	<li>Resuspend in Flow Cytometry Staining Buffer solution and run samples through FACS machine 3,9.</li>
</ol>

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The authors have nothing to disclose.

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 PCR) for our immune cell isolation and routinely get ~2 x 106 cells per isolation. We have used this method to isolate large number of immune cells from the liver and heart (multienzyme digestion for the heart combined with mechanical disruption, data not shown) but are confident that mechanical disruption can be applied to other non-fibrous tissues such as the brain 16.
As with any protocol to isolate immune cells from tissues, adhering to detail is critical for success. If perfusion of the kidneys is critical, then practice is needed in placing the needle into the heart to prevent it from fibrillating or from penetrating too far to the right ventricle. An alternative to putting a needle into the heart is to cannulate the aorta above the renal artery and perfuse the kidneys 8. In addition, there are other suggestions from the authors to make the isolation of immune cells reliable and uniform. The kidneys should be adequately mashed and no large chunks should be visible. If this is not the case, then repetition of the mechanical disruption should solve the problem. The mechanically disrupted tissue should be passed through the right sized filter in order to include cells of all sizes. For example, some macrophage/dendritic cells populations may be excluded if using a 40 &#181;m mesh as opposed to using a 100 &#181;m mesh (macrophage/dendritic cell sizes vary between 10-40 &#181;m). However, on the flip side, more kidney epithelial and other cells could be included in the final analysis changing the percentages of immune cell populations. We believe that being all inclusive increases the representation of underrepresented populations and hence improves the reliability of the prep.
When adding the cell suspension to the density gradient reagent, the cells should be resuspended in no more than 200-300 &#181;l of the Flow Cytometry Staining Buffer solution. Formation of the 36%:72% gradient should be ensured. For this, introduction of the transfer pipette carrying the 72% density gradient reagent into the 36% density gradient reagent should be gentle, should not leave bubbles and a clear demarcation seen between the gradients. As far as possible, centrifugation of cells in density gradient reagent should be done at room temperature. Complete removal of the &#34;cell debris&#34; at the top surface of the gradient after the spin should be ensured. Otherwise, it will interfere with the cell yield and the quality of the prep. Take care to exclude the tiniest cells on the hemocytometer. These are left over RBCs. This is particularly important if the RBC lysis step is omitted. There is a limitation to the method. Not every cell isolated using this method is an immune cell. A large percentage of cells are epithelial cells (includes proximal tubule, distal tubule and glomerulus). However, this limitation can be used to an investigator&#39;s advantage as questions pertaining to non-immune cells can be answered.
To conclude, the method for isolation of immune cells from the kidney presented here is widely applicable and can be adapted by most labs to their research. We believe that this technique can be used for other non-fibrous tissues such as liver and brain as well. Isolation of a large number of immune cells will also help to design more ambitious downstream experiments such as proliferation assays, cytokine secretion assays and isolation of a subset of cells based on surface antigens. Ultimately, we hope that this method will standardize immune cell isolation i.e. avoid various levels of enzymatic digestion of surface antigens.

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><tbody><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&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Electron Microscopy Sciences</td><td>63514-11</td><td></td></tr><tr><td>Analytical flow cytometer</td><td>BD LSR-X20 Fortessa</td><td></td><td></td></tr><tr><td>Percoll&amp;nbsp;</td><td>Sigma</td><td>P1644</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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></tr><tr><td>Fixable Viability Stain</td><td>BD Biosciences&amp;nbsp;</td><td>FVS510,&amp;nbsp; 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)&amp;nbsp; BV421&amp;nbsp;</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></tr><tr><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 &amp;mu;m filter&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Fisherbrand Tubes 15 ml</td><td>Fisher</td><td></td><td>Or equivalent equipment&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Flow Cytometery Staining Buffer Solution</td><td>EBioscience</td><td>00-4222-26</td><td>Or equivalent equipment&amp;nbsp;</td></tr><tr><td>1x RBC Lysis Buffer</td><td>EBioscience</td><td>00-4333-57</td><td>Or equivalent equipment&amp;nbsp;</td></tr></tbody></table>

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 viability stain (FVS510) is used to exclude the dead cells from the analysis (Figure 1, top middle dot plot). CD45, a marker for bone marrow derived hematopoietic cells is typically used to delineate the kidney immune cells and these typically range from 2-18% of the total kidney immune cells depending on the age, sex, strain, inflammatory condition of the mouse (Figure 1, top right dot plot). From the CD45+ bone marrow derived hematopoietic cells, one can either gate on CD3 epsilon as a marker for T-lymphocytes or proceed to CD4+/CD8+ to separate the effector/cytotoxic cells from the bone marrow derived hematopoietic cells (Figure 1, bottom right dot blot). Typically, more CD4+ than CD8+ cells are seen. Further characterization of the CD4+ cells revealed ~8.8% FoxP3+ cells that are likely T-regulatory cells (Figure 1, bottom middle dot plot). Further detailed characterization may include markers such as CD25 and CD127 to distinguish which subset is altered depending on the population of interest. CD127 is largely a marker for FoxP3- cells and this is further dissected out by using CD44 as marker of activation, CD44+CD127lo/- best resemble activated effector cells versus CD44+CD127+ cells that best resemble effector memory cells.
For the macrophage/dendritic cell panel, the initial gates are the same, including the FVS510 based separation of live/dead (Figure 2, top 2nd from left dot plot) and CD45 gates to separate the bone marrow derived hematopoietic cells (top 2nd from right dot plot). Next, the CD45+ cells can be gated several ways. We gated them on Ly6G here to separate neutrophils from the rest of the monocytes i.e. macrophages/dendritic cells, (Figure 2, top right dot plot). Next, Ly6G- cells were gated on CD11b and F4/80 to identify subsets of macrophages and dendritic cells (Figure 2, bottom 2nd from left dot plot). Gating on Ly6C identifies the percentage of infiltrating/inflammatory monocytes/macrophages (bottom 2nd from right histogram) and CCR2 and CX3CR1 identifies the macrophages that were recruited (Figure 2, bottom right dot plot). Bottom left most panel identifies most cells as dendritic cells, a lower portion of which were recruited via CCR2 expression.
<img alt="Figure 1" src="/files/ftp_upload/54368/54368fig1.jpg" /> 
Figure 1: Representative Dot Plots Displaying T-Lymphocyte Subset Isolation. Top left is a typical forward (FSC) and side (SSC) scatter profile of kidney immune cells in the Flo Jo software. Top middle panel is gated on a Fixable Viability Stain (FVS510) and the live population on the left is then gated on CD45 for bone marrow derived immune cells. CD45+ cells are gated for CD4 (effector) and CD8 (cytotoxic) cells (Lower right panel). Middle panel shows the number of CD4+ cells that are FoxP3+ (T regulatory cells). Lower left panel attempts to tease out how many of the CD4+Foxp3- cells are activated effector CD44+CD127lo/- versus memory effector CD44+CD127+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54368/54368fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54368/54368fig2.jpg" /> 
Figure 2: Representative Dot Plots Displaying Monocyte-Cell Subset Isolation. Again, top left is a typical forward (FSC) and side (SSC) scatter profile of kidney immune cells in the Flo Jo software. Top 2nd from left dot plot is gated on a Fixable Viability Stain (FVS510) and the live population on the left is then gated on CD45 for bone marrow derived immune cells (Top 2nd from right dot plot). CD45+ cells are gated for Ly6G (neutrophils, top right dot plot) in order to separate for macrophages and dendritic cells. Gating on Ly6C identifies the percentage of infiltrating/inflammatory monocytes/macrophages (bottom 2nd from right histogram) and CCR2 and CX3CR1 identifies the macrophages that were recruited (Figure 2, bottom right dot plot). Bottom left most panel identifies most cells as dendritic cells, a lower portion of which were recruited via CCR2 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54368/54368fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Reagents and Equipment Needed for Immune Cell Isolation From the Kidney. See Materials Table.
Table 2: Table Depicting total Cells and CD45+ Isolated in the Corresponding Prep. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54368/Table2.xlsx" target="_blank">Please click here to download this file.</a>

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 ...

reliable-and-high-efficiency-extraction-of-kidney-immune-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.3K Views.  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.

Video: Reliable and High Efficiency Extraction of Kidney Immune Cells

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioengineering

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Medicine

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

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

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

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

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

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Biology

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

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Isolation of Double Negative αβ T Cells from the Kidney