(C2C12 myoblasts)
From the Laboratory of Helen M. Blau, Stanford University
T. Wehrman, 12/2002
Preparation of Staining Solution:
• Solution A: 1mM CCF2/AM dissolved in dry DMSO
• Solution B: 100 mg/ml Pluronic acid in DMSO containing 0.1% acetic acid (provided in kit)
Solution A should be aliquoted and stored at -20°, to prevent repeated freeze/thaw cycles. Stored in this manner the substrate is stable for at least one year.
Just before staining the cells mix 5 ul of solution A to 20 ul of solution B. This mixture is then added to 2.5 ml DMEM (no serum). Probenecid should be added to the resulting mixture at a final concentration of 2.5 mM.
Staining Protocol:
Trypsinize cells and wash once on growth medium (20%FBS DMEM P/S)
Resuspend the cell pellet in prepared staining solution.
Incubate at room temperature 1-2 hours.
Optional: pellet cells to remove staining solution, resuspend in PBS 5% FBS and place on ice until the FACS analysis.
Excitation should be made by a uv or Krypton laser. Typical signals should be observed in the cascade yellow and cascade blue channels.