A Technique to Isolate Satellite Cells From Rat Head Muscle Tissue
Transcrição
To isolate the satellite cells, transfer each muscle into individual wells of a six-well plate, and cut the tissues into small 2-millimeter pieces, taking care not to mince the muscles too much. Next, carefully incubate the tissue fragments in 2.5 milliliters of 0.1% pronase solution per well at 37 degrees Celsius for 60 minutes, with gentle shaking, every 20 minutes.
When the fiber bundles exhibit a loosened appearance, add 2.5 milliliters of DMEM, supplemented with horse serum and antibiotics to each well, and transfer the digested tissues to individual 15-milliliter conical tubes. Spin down the tissue slurries, and decant the supernatants. Then, add 5 milliliters of medium to each tube, and homogenize the tissue with a 10-milliliter plastic pipette.
Spin down the tissues again and transfer the supernatants into new 15-milliliter conical tubes. Then, add 5 more milliliters of medium to the tubes, and triturate the tissue pieces again until the fragments pass easily through the pipette.
After another centrifugation, pool the supernatants into the appropriate corresponding tubes, and filter the cell suspensions through 40-micrometer strainers into individual 50-milliliter tubes. Wash the strainers with 1 milliliter of DMEM for maximal cell recovery. Then, spin down the cells. Resuspend the pellets in fresh medium and count the cells.
To culture the isolated satellite cells, next, dilute the cell suspensions to 1.5 x 103 cells per 10 microliters of culture medium. Then, add 10 microliters of cells onto each previously prepared extracellular matrix gel spot and incubate the gels at 37 degrees Celsius. After six hours, carefully add 400 microliters of culture medium to each gel spot, and return the slides to the incubator. After three days, initiate the differentiation of the cells with the addition of the appropriate culture medium.
