CD8+T cells carry distinct features in different tissue, as a critical example of non-mucosal and non-lymphoid tissue. It is essential to directly characterize kidney-resident cells to fully understand T-cell biology. Here we will demonstrate our convenient method to isolate kidney-resident CD8+T cells accommodating subsequent flow cytometry analysis.
This method could provide insight into the studies of effector and memory CD8+T cells generated after infections as well as autoimmune responses. The procedure will be performed by Dr.Chaoyu Ma, a research scientist for my laboratory. Begin by diluting biotin anti CD8 alpha antibody to 15 micrograms per milliliter in PBS, making sure that there is enough to administer 200 microliters to each mouse.
Prior to injection, heat the tail vein of the mouse with an overhead heat lamp for five to 10 minutes to dilate it. Draw 200 microliters of the pre diluted antibody mix into a 28-gauge insulin syringe and remove any air bubbles by moving the piston up and down. Bend the needle to create a 150-degree angle between the needle and the syringe, bevel up, so that the needle is parallel to the tail vein.
Restrain the mouse with a rodent restrainer and spray the tail with 70%ethanol to make the vein clearly visible. Hold the tail at the distal end with the thumb and middle fingers of the non-dominant hand, then place the index finger underneath the injection site. With the dominant hand insert the needle into the vein in parallel towards the direction of the heart and slowly inject 200 microliters of the antibody.
Remove the needle and gently compress the injection site until bleeding stops. After euthanizing the mouse, dissect the kidney with scissors and transfer it to a 1.5-milliliter micro centrifuge tube. Leave the samples on ice until further processing.
Prepare six-well plates with three milliliters of digestion solution in each well, using one well per mouse, and store them on ice. Add 300 microliters of digestion solution into each sample tube and mince the kidney samples with a straight spring scissor. Transfer the minced kidney samples to the six-well plates with the digestion solution.
Incubate the samples at 37 degrees Celsius with gentle rocking for 45 minutes, then homogenize the tissue with the plunger flange of a three-milliliter syringe and transfer it to 15-milliliter conical tubes. Spin the samples down at 500 times g and four degrees Celsius for five minutes. Remove the supernatant and resuspend the pellet in three milliliters of RPMI with 10%FCS.
Spin down the sample at 500 times g and four degrees Celsius for five minutes, then remove the supernatant and resuspend the cell pellet with five milliliters of 44%density gradient medium and RPMI mix. Put the tip of a three-milliliter pipette containing three milliliters of 67%density gradient medium and PBS directly to the bottom of each tube and slowly release the solution so that the heavy solution forms a distinct layer at the bottom. Spin the samples at 900 times g for 20 minutes with reduced accelerator and brake settings, then carefully remove the tubes from the centrifuge without disturbing the layers.
Remove the top layer with a transfer pipette without touching the lymphocyte layer and transfer the lymphocyte layer to a new 15-milliliter tube. Fill the tube with PBS and 5%FCS, then mix by slowly inverting the tube four to six times. After centrifuging the samples at 500 times G and four degrees Celsius for five minutes, remove the supernatant and resuspend the cell pellet with 500 microliters of complete RPMI medium.
The cells are now ready for flow cytometry staining. Even after density centrifugation-mediated lymphocyte enrichment, it was very common to see a large portion of non lymphocytes in the final product. However, after gaining on live lymphocytes, CD8+T lymphocytes were easy to identify.
As expected, only extra-vascular CD8+T cells efficiently acquired tissue resident memory T-cell phenotypes, such as upregulation of CD69 and downregulation of Ly6C. In contrast to the infected mice, the vast majority of CD8+T cells isolated from young naive mice housed in the SPF facility were labeled with CD8 alpha antibody and therefore belonged to the intravascular compartment. This technique has greatly accelerated the investigation of tissue-resident immune cells in densely vascularized organs.
