Reagents

• Appropriate culture media (i.e. cRPMI for T cell lines, cDMEM for attaching cells) warmed in 37°C waterbath.

• cRPMI = RPMI + 10% FBS+ L-Glut + P/S.

• cDMEM = DMEM + 10% FBS+ L-Glut + P/S + HEPES

• Indo-1 AM, 1 mg/ml in DMSO, store at -20°C, Calbiochem

• Ionomycin, 1 mg/ml in DMSO, store at -20°C, Calbiochem

• Dimethyl sulfoxide (DMSO; Sigma), 10% bleach or ethanol.

• Antibiotics/proteins/growth factors as needed. Monoclonal antibodies must be azide-free. Dialyze against PBS overnight.

• 5 ml FACS tubes, Falcon

Protocol

Indo-1 Loading

1. Count cells and adjust cell concentration to 1 x 10⁷/ml in culture medium.

2. Add 7 µl* of stock Indo-1 per ml of cells and wrap the tube in foil.

3. Incubate the cells with Indo-1 for 45 minutes in a 37°C water bath. Gently mix the loading cells every 10 to 15 minutes to ensure even loading.

4. Wash the cells once with culture medium at 1200 rpm for 8 minutes.

5. Gently resuspend Indo-loaded cells at 5 x 10⁶/ml in culture medium.

6. Store the Indo-loaded cells wrapped in foil at room temperature until the cells can be run on the LSR. Let cells rest for ~15 minutes before starting. Indo-loaded cells should never be placed on ice, because this will inhibit [Ca²⁺]_i signaling.

Sample Running

1. Before starting Indo-1 experiment, turn on circulating waterbath, so it can reach 37°C by the start of your run.

2. Turn LSR on 30 minutes before run. Plug in UV. Make sure the proper filters are in place for running Indo-1/AM: FL5 detector 400BP40, FL4 detector, 510BP20.

3. LSR files for screens, settings and data are found in LSR CAF Stuff user files. Use screen ABW Calcium Flux and settings ABW Calcium. Save data in current week folder.

4. Prewarm 0.8 ml culture medium in several 5 ml FACS tubes to 37°C.

5. Add 0.2 ml Indo-loaded cells (1 x 10⁶)/tube and warm to 37°C for 3 to 5 minutes before running on the LSR.

6. Run cells (400 cells/second) and establish a baseline of [Ca²⁺]_i for 40 to 60 seconds before adding stimulant.

7. FL4-H: M1 gate should contain 80% of the Indo-loaded cells (between 600-800 on X-axis). FL5-H/FL4-H ratio should be just below 200 on the Y-axis.

8. Add 10-50 µl of stimulant and collect for another 5 minutes (this may vary depending on the kinetics of the response).

9. Test ionomycin response first with 5 µl stock ionomycin. This is the maximum stimulation and tests proper loading of the cells (all cells should respond). If indo-loading is not good, the other samples should not be run.

10. After running the ionomycin samples (or other strong stimulants) be sure to flush the lines 1 minute with DMSO or 10% bleach or ethanol, then 1 minute with culture medium to bind any residual ionomycin.

11. Click on pause and save. Print graph.

12. Analysis of data with FlowJo software.

• Indo-1 AM is a fluoescent dye which, as a free ester, absorbs at 349nm. Chelated to calcium it emits at 405nm. It enters cells irreversibly due to cytosolic esterases.

• The optimum concentration of Indo-1 should be determined empirically, but must not exceed 5 mM. At higher concentrations the fraction of Indo-1 that binds Ca2+ will be decreased, resulting in a color change that is less apparent.

• The molecular weight of Indo-1 AM ester is 1009.9 g/M; a 1 mg/ml stock is almost exactly 1 mM.