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. 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 °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.
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 removes the blood which may interfere with the interpretation of data. Hence, if possible we always perfuse the organs.
2. Dissection of Kidneys
3. Homogenization of Kidneys
4. Gradient Centrifugation
5. Optional (Cell Labeling)
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.
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+. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to download this file.
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 µm mesh as opposed to using a 100 µm mesh (macrophage/dendritic cell sizes vary between 10-40 µm). 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 µl 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 "cell debris" 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'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.
The authors have nothing to disclose.
This work is supported by a Research Grant from Dialysis Clinics Inc. and from the University of Missouri Research Board Grant.
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 |