Ratiometric Fluo-3/Fluo-4 and Fura-RED Protocols

1. Pellet cells and adjust to 10 x 10⁶/ml.

2. Dissolve 50 mg freeze-dried aliquot of Fluo-3/4 (Invitrogen, Cat. No. F1242, F14201) with 100 ml DMSO.

3. Dissolve 50 mg freeze-dried aliquot of Fura-RED (Invitrogen, Cat. No. F3021) with 100 ml DMSO.

4. Incubate lymphocytes with 1-2 mM Fluo-3/4 and 8 mM Fura-RED at 37⁰C for 45 min.

5. Wash twice in calcium free PBS.

6. Adjust cells to 1 x 10⁶/ml in Falcon 5ml tubes.

7. Run flow cytometer with 530 nm and 600 nm channel on linear against the Time parameter with the ratio set with 530 nm channel as numerator and 600nm channel as denominator.