Indo-1 Loading Protocols

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

2. Dissolve 50 mg freeze-dried aliquot of Indo-1 (Invitrogen, Cat. No. I1223) with 50 ml DMSO.

3. Incubate lymphocytes with 1-2 mM Indo-1, Granulocytes/Monocytes with 2-3 mM Indo-1 and dendritic cells 4-5 mM Indo-1at 37⁰C for 45 min.

4. Wash twice in calcium free PBS.

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

6. Run flow cytometer with 530 nm and 440 nm channel on linear with a 405 nm dichroic mirror to split the indo-1 signal. The ratio parameter set with 530 nm channel as denominator and 440 nm channel as numerator.