Yes, Resul Kiymaz.

This flow cytometer can perform intracellular calcium ion flow test, Ca2+ concentration determination, and intracellular pH measurement.

Some suggestions:

1) Since you are using fibroblasts, it is recommended to use DMEM or MEM culture medium without phenol red (phenol red may interfere with the fluorescent signal). The density of the cells after resuspension is adjusted to 1×10⁶ cells/mL.

2) We also provide Fluo-4 AM (Cat.NO.: HY-101896), which is suitable for flow cytometry and has a detailed protocol. If you are interested, you can download the PDF on the official website:

https://www.medchemexpress.com/Fluo-4_AM.html

Tips:

The working solution concentration of Fluo-4 AM (λex=494 nm, λem=516 nm) is recommended to be 1 μM, and it should be prepared and used immediately.

Mix the cell suspension with the Fluo-4 AM working solution at a ratio of 1:1 and incubate at 37℃ in the dark for 30-45 min. 300 g, centrifuge for 5 min, discard the supernatant, wash twice with preheated HBSS or calcium-free PBS.

Finally, resuspend in 1 mL detection buffer (containing 2 mM Ca²⁺, 1 mM Mg²⁺), keep at 37℃ for use.

3) If there are problems such as weak or no fluorescence signal, high background fluorescence, and large fluctuations in calcium signal, it is mainly caused by incorrect Fluo-4 AM incubation time and post-incubation washing operations. We can discuss specific issues. Welcome to contact me again.

Hello, Resul Kiymaz.

I don't know if you have already performed this experiment.

We discussed the precautions and hope to continue to give you feedback:

In general, it is not recommended to use flow cytometry for Measure Calcium Content.

Because the calcium ion concentration in cells is relatively low under normal physiological conditions, most dyes may not be detected.

After stimulation/drug treatment, the intracellular calcium ion concentration will change; this is an instantaneous change, measured in seconds or even milliseconds (you can learn about calcium sparks).

However, flow cytometry is a complex staining and loading process after the cells are processed (stimulated), and there is a time delay in the middle, so there will be errors in the detection. It is recommended to refer to papers in the same research direction to determine whether it is reasonable for you to use this experimental method now.

Hello,

I have never measured Ca2+ in fibroblast, but I've used the FACSCalibur to measure Ca2+ signalling in human monocytes, neutrophils and eosinophils. I use FuraRed and Fluo-3 together. It works really well.