Dear Shir Kn,

If you are having background problems, you could firstly check the purity of reagents. Then, review the followed protocol, as you can reduce the concentration of second antibody while increasing its incubation and washing times. Also, do you have a negative control? That would help you to confirm what is important for your study. In addition to the negative control, there’s two strategies to determine background noise: 1) Check the stronger length of excitation or emission of your autofluorescence and test the fluorescent label in a different wavelength [1]. 2) Also, you can remove background data interference by chelating all background Ca2+ with a chelate agent as EGTA or BAPTA. BAPTA has been related to more specificity for Ca2+ than EGTA.

With respect to ionomycin, there is several reports of its functionality in epithelial cells. Thus, this problems could be related to concentrations, protocols or similar, but not cell type. To improve the usage of ionomycin, you can check its activity at 37°C, as it is closer to physiological conditions and it is reported to improve ionomycin activity. Additionally, depending on your project's objective, if you are looking for Ca2+ uptake you can also consider Ca2+ ionophore A-23187; contrary, if you are trying to accumulate calcium, you can view thapsigargin and cyclopiazonic. Another strategy could be the mRNA expression analyses of ER stress (Xbp-1) as it is reported that it shows good reads of calcium concentrations [2].

Also, the Ca2+ changes could be better observed if the reagents has optimal conditions. For that, It is recommended to use a fresh dye for every analysis, because Fluo-4 is affected at low temperatures. The ionomycin has to be aliquot and changed every 3 months once in solution, as well as the EGTA (if selected), as they have been related to misfunction when refrozen [3].

Hope it works!

[1]. Zhang, R. et al. (2018). Macroscale fluorescence imaging against autofluorescence under ambient light. Light Sci Appl 7, 97.

[2]. He, Y. et al. (2010). Emerging roles for XBP1, a sUPeR transcription factor. Gene expression, 15(1), 13–25.

[3]. Cell Signaling Technology. (2014). Ionomycin, Calcium Salt, 9995. Cell Signaling Technology, Inc.

I suggest using DPBS without Calcium and Magnesium in place of PBS for Flow

María Durán Thank you for the detailed response and suggestions!

As for the storage conditions, I ordered Fluo4 which is already dissolved in DMSO, and manufacturer instructions were to keep it at -20C, so that's what I'm doing. Did you mean I should divide it into smaller aliquots to avoid freeze-thaw cycles?

I will also add a chelating agent and a negative control as you suggested.

As for the ionomycin, I have since solved the problem by trying much higher concentrations, which seemed to work even on the epithelial cells.

However, the spike in Fluo4 staining in the epithelial cells, even with the high ionomycin concentration, was still relatively small compared to the very high basal level.

To solve this I tried reducing the concentration of Fluo4, as well as the incubation time and temperature of the Fluo4 loading step, but the problem persisted (there was a slight reduction in the basal Fluo4 staining but it was still high).

Eventually I hypothesized that the Fluo4 might be compartmentalized in organelles in the epithelial cells, and this is why I see high background and not much increase when cytosolic calcium increases, as I understand this might be an issue. I did mild permeabilization of the plasma membrane and indeed found that the T cells lost the Fluo4 staining (meaning it was in the cytosol) but the epithelial cells haven't lost it (meaning it was stored in organelles). So I now believe my problem is compartmentalization.

Do you have any idea how I can solve this?

Utkarsh Reddy Thank you for your suggestion! But just a followup question- if I use PBS without calcium will I still see an increase with my positive control (ionomycin)? As I understand ionomycin causes an extracellular calcium influx, which will not happen if there is no extracellular calcium...

Many thanks again!