I've been trying to check the calcium influx in NIH-3T3 cells transfected with a GPCR. The cells are incubated in fura 2AM in 5M concentration in serum free cell culture medium for 30 min at 37°C. I check the viability of the cells before incubating them in fura 2. But after incubation, the amount of live cells (checked by trypan blue) go down and as I proceed with estimation of Calcium ion on flourimeter, the number of live cells almost becomes nil as a result of which I dont get any signal. I incubate the cells in KRH buffer during the experiment. Can anyone suggest me the reason of cell death?

Calcium Ion Estimation

Hello joyi:


You may want to take a look at the following reference...

Takahiro Taira, Yoshiro Saito, Takeshi Niki, Sanae M.M. Iguchi-Ariga, Kazuhiko Takahashi & Hiroyoshi Ariga. DJ-1 has a role in antioxidative stress to prevent cell death. EMBO reports 2004, Vol 5 - No 2: 213-218.

Using siRNA against DJ-1 which was transfected into a SH-SY5Y cell line, the authors were able to demonstrate that DJ-1 has a role in oxidative-stress induced cell death. They also indicated that "The same activity of DJ-1 against cell death was also observed in mouse NIH3T3 cells" but the data was not shown.

I realize that this doesn't specifically answer your questions, but hopefully this can point you in the right direction.

Good luck!

Hey,

I need to estimate calcium concentration in plant cell extract. Would you be able to give me some guidelines/procedures? I will be extremely thankful!

best,

MSM

The easiest way to obtain relative Ca2+ levels would be a fluorimetric assay using Fluo-3 or Fluo-4 (you do not need the cell permeable ester version, designated Fluo-3AM etc) from either Sigma or Molecular Probes (Invitrogen). You will need to titre out a range from ~0.5 um - 10 uM to find saturating levels, and measure flurescence after ~20 min incubation. If you can prepare a Ca2+ std curve, you can have a semi-quant assay.