Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney
Transcript
After freeing the kidneys as outlined in the text manuscript, hold the kidney with the surgical forceps and use another pair of forceps to pull off the renal capsules. After this, place the kidneys into the wells of a 6-well culture plate that each contain 2 milliliters of HBSS and place the culture plate on ice.
Next, transfer the kidneys to a 100-millimeter Petri dish and use two scalpels to mince them into small pieces that are approximately 1 to 2 millimeters. Keep the minced kidney pieces wet with HBSS.
Next, place the kidney pieces on top of a 300-micrometer metal sieve and press the kidney through the sieve using the plunger from a 20-milliliter syringe. Repeatedly rinse the sieve with HBSS in between and use a serological pipette to collect the flow-through and transfer it to a clean Petri dish.
Use a scalpel to scrape off everything that remains in the bottom of the sieve and transfer it to the collected kidney homogenate. Rinse the kidney homogenate through a 75- and a 53-micrometer sieve with HBSS. Then, wash both sieves with HBSS to remove all of the smaller structures.
Collect the kidney structures and material that remain in the sieves by washing the upper surface of each with DMEM supplemented with 20% fetal calf serum and transfer the material to the wells of a 6-well ultra-low attachment microplate.
Bring the ultra-low attachment microplate to an inverted light microscope and use a 20-microliter pipette to collect single encapsulated and/or decapsulated glomeruli.
