Dissolve Indo-1 (Molecular Probes catalog No. I1226) in DMSO to a final concentration of 2 mg/ml (25 ul of DMSO into 50 ug of Indo-1).
Mix quantities of probenecid (Sigma Catalog No. P8761) and water to yield a 100 mM solution. Weigh 285 mg of probenecid, add 8 ml of dH2O, then add 1 M NaOH until dissolved. The pH should be between 9 and 10. Store for 1 month at room temperature.
Dissolve Ionomycin (Calbiochem Catalog No. 407950) in 100% ethanol for a final concentration of 1 mg/ml. Store at –20°C. This is used as a control for calcium flux.
Cell Loading Media (CLM) is Hanks Balanced Salt Solution (HBSS) containing 1 mM calcium, 1 mM magnesium, 1% FBS or 0.5% BSA and can be stored up to 1 month at 4°C.
Sample Preparation:
In a 15 ml tube, suspend the cells in CLM at a concentration of 107 cells per ml.
Add 40 ul of a 100 mM probenecid stock to 1 ml of cells for a final concentration of 4 mM.
Add 2 ul of a 2 mg/ml Indo-1 stock to 1 ml of cells for a final concentration of 2 ug/ml.
Incubate the cells with Indo-1 AM and probenecid for 30 minutes at 37°C.
Wash the cells twice in CLM.
Resuspend the cells in CLM at a concentration of 107 cells per ml.
Rest cells for 15 minutes before acquiring any data.
100 ul (1 x 106 cells total) of the Indo-1 loaded cells can be used for additional cell surface staining.
Cell Surface Staining (if required):
Add the appropriate mAb to 1 x 106 cells (100 ul of the Indo-1 loaded cells).
Incubate for 20 minutes at room temperature.
Wash once in CLM.
Cells can be held at 4°C for several hours. At the time of analysis, cells should be warmed up to 37°C. Each tube should be warmed separately just before running.
Instrument Set up:
Verify filter configuration: 530/30 PMT A (Indo-1 blue) and 440/40 PMT B (Indo-1 violet) on the UV trigon.
Under the ratio tab click add and choose Indo-1 (violet) for numerator and Indo-1 (blue) for the denominator.
Create the following plots on a Global Worksheet: FSC vs. SSC, FITC vs PE, Indo-1 (violet) vs Indo-1 (blue) and Time vs Ratio Indo-1 (violet)/Indo-1 (blue).