The following protocol, our most widely-used method for intracellular calcium flux determination, employs the probe Indo-1. Indo-1 requires UV (350nm) excitation, and has a dual emission pattern: dye bound to free intracellular calcium will emit at 395nm, while the dye alone will emit at 525nm. Data analysis involves assessing the ratio of the emission wavelengths vs. time of acquisition which encompasses the baseline reading, the injection of agonist, and the return to baseline fluorescence levels. The core facility has software for analysis of kinetic experiments (Multitime, Phoenix Flow Systems) to obtain detailed analysis of the calcium responses.
Technical Considerations/Troubleshooting:
- Cell condition and number: As with any cell function assay, cells should be in good condition and at baseline function level (i.e., not activated). The starting number of cells per iteration will depend on the length of sample collection required–i.e. the time for cells to flux and return to baseline. Recommend starting with at least 5×105 cells.
- Medium: The medium in which the cells are suspended for dye loading and acquisition can be the cell culture medium your cells utilize with the caveat that it must contain calcium.
- Temperature: Samples should be equilibrated at 37°C for approximately 10 minutes prior to data acquisition on the flow cytometer.
- Dye Concentration: Similar to most flow cytometry protocols, the final concentration of Indo-1 utilized in any given system should be determined by titration. Typical concentrations utilized range from 1uM to 10uM. Overloading the cells can blunt the calcium response.
- Controls: It is advisable to have controls and test each variable in the experiment-i.e. agonist, responders vs. non-responders, etc. Calcium ionophore can be a useful indicator of a positive response with the caveat that the responses are several orders of magnitude above a normal physiologic response, i.e. it is not a sensitive trouble-shooting tool.
- Calibration: While not detailed in the attached procedure, calcium responses in a given system can be calibrated. See core facility personnel if interested.