Hi everyone, I'm currently working with Fluo-4 AM for calcium imaging in HeLa cells, but I'm running into a few issues and would really appreciate your suggestions:

• Problems:

• Protocol: *with light protection throughout

*No ATP or other stimulants were used. Pluronic F-127 was also not included, as it is currently not available in our lab.

• Questions:

1. About weak signals and high background:

(1) Is Pluronic F-127 essential for efficient dye loading in HeLa cells? Would it significantly improve the signal intensity or reduce background fluorescence?

(2) Additionally, what other methods or tips would you recommend to reduce background fluorescence and improve the signal-to-noise ratio in Fluo-4 calcium imaging experiments?

2. About positive control:

(1) I forgot to include a positive control in this experiment. Since I can see faint cell signals (especially at longer exposure times), is it still necessary to include a positive control (e.g., ionomycin or ATP) to validate dye loading efficiency?

(2) If a positive control is needed, could you suggest a practical and effective compound, especially considering that we currently don’t have calcium agonists like ATP or ionomycin in the lab?

Thank you very much for your time and any advice you can offer! Your insights would be really helpful as I troubleshoot this.