Fluo-3 and Fluo-4 Protocols

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

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

3. Or dissolve 50 mg freeze-dried aliquot of Fluo-4 (Invitrogen, Cat. No. F14201) with 100 ml DMSO.

4. Incubate lymphocytes with 1-2 mM Fluo-3/4, granulocytes 2-3 mM, dendritic cells at 4 mM 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 cell samples with 530 nm channel in linear mode against time.