| Phosphate-free DMEM (1×) |
16.3–18.3 mL (depending on serum concentration) |
1× |
| Dialyzed calf serum |
0–2 mL |
0%–10% |
| l-Glutamine (100×, 200 mm) |
0.2 mL |
2 mm |
| HEPES-KOH (pH 7.4, 1 m)
|
0.5 mL |
25 mm |
| [32P]Orthophosphate (10 mCi/mL)
|
1.0 mL |
500 µCi/mL |
| Add all reagents, except [32P]orthophosphate, under sterile conditions in the tissue culture hood and warm in water bath set to 37°C. Add [32P]orthophosphate to medium on the laboratory bench to avoid contamination of the tissue culture hood just before adding Labeling
medium to cells.
|
