Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells
Transcrição
To process the harvested kidneys, first, remove the renal capsules and medulla. Then, mince both kidneys into tiny pieces and incubate them in 10 milliliters of a digestion buffer in a 37 degrees Celsius oven with gentle rotation for 5 minutes.
After the digestion, remove any undigested tissues by passing the buffer through a 70-micron filter. After collecting the digested tissue, add 10 milliliters of culture media to stop the digestion. Next, centrifuge the filtered tissue suspension at 50 x g for 5 minutes to pellet the tubular cells. Then, transfer the supernatant to a new tube, add 5 milliliters of culture media, and repeat the centrifugation to collect any remaining tubular cells.
Now, resuspend the first pellet in 20 milliliters of culture media and centrifuge it at 50 x g for 5 minutes to further purify the collected cells. Dispose of the supernatant. Then, resuspend both pellets in 10 milliliters of culture medium. Next, count the living cells using Trypan Blue staining. Then, seed up to 10 million cells onto a 60-millimeter collagen-coated dish and let the tubular cells attach overnight.
