Hello Guys,
Can someone provide me with a simple protocol to measure intracellular calcium ? I want to isolate intestinal epithelial cells from mice and measure Calcium level ? Can we detect calcium by IF staining ?
Thanks in advance
Dear Doaa,
to measure Calcium levels you need to use a fluorescent probe. Depending on the expected level of calcium and on the technique you want to use to detect fluorescence (basically fluorimeter or cytofluorimeter) you can choose a probe from a wide list. I suggest you to consider as a reference the excellent guide of Thermofisher (https://www.thermofisher.com/it/en/home/references/molecular-probes-the-handbook.html).
Anyway, I have no experience in measuring calcium level in isolated cells, but I used Fluo4 AM probe with several cells lines (mouse epithelial, mouse and human neuroblastoma) and it worked pretty well. I grew cells in multiwell screening plate, treated them with the probe and measured the calcium level with a fluorimeter equipped with a plate reader (the protocol is pretty easy).
Good luck!
Giulia
Incubation steps are quite long: first you have to incubate cells with the probe (in serum-free medium!) for 15 to 60 minutes, then wash and incubate again for 30 minutes with just the medium to allow complete probe de-esterification. For the first incubation in my experience 40 minutes worked well.
In my experience two points deserve special attention:
- calcium probes may bind various heavy metal cations with, sometimes, higher affinity than calcium. In my work that was a huge problem because I was testing effects upon Cd treatment. I decided to use Fluo-4 because is the one with best calcium vs cadmium rate affinity. In the Thermofisher guide affinities for several cations are tested and compared between the different probes.
- if you use the multiwell plate reader you need to find a way to quantify the number of cells that grew in each well. You can detach and simply count them, or you need to find another convenient way.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining