This is the protocol I have been using (and struggling with)
-prior to treatment with 2.5 uM Thapsigargin (A SERCA blocker) or 1uM Ionomycin (a calcium ionophore), the cells are starved for 24 hours (DMEM-FBS)
-treatment is carried out for 20 mins for Thapsigargin/5 mins for Ionomycin @37 degrees and followed by lysis with triton glycerol buffer.
I am using this treatment to look at potential protein-protein interaction on intracellular calcium increase.
To verify if there is calcium increase, I check for phosphorylation of Camkii in the lysates and observe no change.
Any inputs will be highly appreciated.
I'm not sure if you need to starve the cells as a per-treatment as that may cause other unwanted side effects. We usually use the calcium ionophore A23187, thapsi up to 5uM- 3-6hrs or tunicamycin (1ug/mL)- 3-6hrs to increase calcium levels. mRNA expression to measure ER stress (Xbp-1) is usually a good read out for calcium levels. Also I hear Rhodamine – 2 is a good way to measure intracellular calcium (FACS or Immunofluorescence).
I've measure p-CamKII in HEK293 cells but the activation doesn't look great sometimes. Good Luck!
what not use fura-2 analyses to determine if Ca levels are increasing. Any purinergic agonist such as ATP should do it.
The SERCA inhibitor may be a better choice than ionomycin to induce mild and prolonged elevations of intracellular calcium. However I am primarily working with neuronal cells, where 2-2.5uM Thapsigargin proved reliably effictive in raising intracellular calcium (measured by OregonGreenBAPTA-Fluorescence). This dose was even quite well tolerated for prolonged incubation periods (overnight). As a negative control you may consider to reduce intracellular calcium by chelation. I personally prefer BAPTA/AM as a calcium "sponge".
Best regards and good luck!
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