Advice needed on what kits are best for the measurement of intracellular calcium. Any help will be much appreciated. I have been looking at the FLIPR calcium assay kits from molecular devices. Does anyone have experience with these?
Thank you
Hi Roksana,
I frequently use cell permeant Fura-2, AM for intracellular calcium imaging using ratiometry fluorescence microscopy. It is a very widely used technique for precisely measuring intracellular/cytoplasmic calcium in live cell in static or perfusion system. We have a nice set up with temp controlled stage attached to the Nicon Eclipse microscope attached to a CCD camera. We have a shutter instrument attached which allows the excitation wave lengths (340 and 38 nm) sequentally and capture the image at an emission wavelength of 515nm. We also have an InCyte-IM2 (InCyt Im2™ Imaging System, Cincinnati, OH, USA) software that converts the ratiometric signal into an actual calcium read out if the system is pre-calibrated against a series of known calcium using Fura-2 acid form or Fura-2, AM in cells sitting on a glass cover slip or glass bottomed 35mm rimless petri dishes.
Post calibration at the end of each experiment can be done using ionomycin and EGTA treatment to maximize the intracellular calcium.
I prefer using pre-calibrated standards for the experiments performed in the same day using a kit (Fura-2 Calcium Imaging Calibration Kit (Zero to 10 mM CaEGTA, 50 µM Fura-2, Life Technologies, Cat # F-6774) and for the probe loading in cell I use Fura-2, AM, cell permeant (Life Technologies, Cat#F-1201), stock prepared in Pluronic F-127 (20% Solution in DMSO) (Life Technologies, Cat# P-3000MP). Final working loading concentration 3-5uM for 30 min to 1h, with washing and deesterification for another 10-15 min.
We have published series of data in different good journals. I am sharing a representative publication as a ready reference.
I , myself never used FLIPR calcium assay kits so I can not comment anything on it.
Hope that helps!!!
@Roksana,
There is a typo:
The corrected excitation wave lengths are 340 and 380 nm.
Sorry for that.
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