Calcein AM Loading Protocol

1. Pellet cells and adjust to 1-10 x 10⁶/ml, after induction of apoptosis.

2. Dissolve 50 mg freeze-dried aliquot of Calcein AM (Invitrogen, Cat. No.M34153) in DMSO according to the manufacturers instructions.

3. Incubate cells in:- Tube 1 with 100 nM Calcein AM, at 37⁰C for 15 min.

4. Incubate cells in:- Tube 2 - Calcein AM plus 0.4 mM Cobalt Chloride, at 37⁰C for 15 min.

5. Incubate cells in:- Tube 3 – Calcein plus 0.4 mM Cobalt Chloride and 500 nM ionomycin, at 37⁰C for 15 min.

6. Wash in PBS

7. Collect 10,000 events from the three tubes with 530 nm channel.