45Ca2+ uptake study in PC12 cells

45Ca2+ uptake study in PC12 cells

1. Plate the cells on to the poly-D-Lysine-coated six well plates 2 days prior to study.

2. Rinse the cells with 1 ml secretion buffer (150 mM NaCl, 5mM KCl, 2mM CaCl2, 10 mM Hepes, pH 7.4) every 15 minutes for 1 hour at 37^oC.

3. Add 45Ca2+ (2 µCi/ml) to Ca2+ -free buffer (secretion buffer without 2 mM CaCl2), and drugs to the labelled buffer at 37^oC.

4. Incubate the cells with labelled buffer with different conditions for 1 minute at room temperature.

5. After 1 minute dump the solution and add 2 ml of ice cold Ca2+ -free buffer with 2 mM LaCl3 and 2 mM EGTA.

6. Wash the cells 6 times with ice cold Ca2+ -free buffer with LaCl3 and EGTA.

7. Add 1 ml of cell lysis buffer to each well.

8. Collect the cell lysate after 30 minutes and count in a beta-counter using

program for 14C.

9. The data are expressed as dpm/well.

Source of the reagents: 45Ca2+ (Dupont) (Calcium chloride in water; specific activity: 25.92 mCi/mg).