Mullins Molecular Retrovirology Lab

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

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

8. 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.

9. 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.

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

11. 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.

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

13. 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.

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

15. 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.

16. 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.

17. Click on pause and save. Print graph.

18. 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.