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.
