The protocol below provides information on how to measure free cytosolic calcium concentration ([Ca2+]i) in CHO-K1 cells using Fura-2 AM.
Materials and Reagents
- Ca2+ indicator Fura-2 AM
- Phenol red-free DMEM
Equipment and Software
- Nikon Eclipse TE200-E microscope (or equivalent)
- MetaFluor Software (or equivalent)
- Plate 50000 CHO-K1 cells/ml onto 25 mm round glass coverslips for 24 h.
- The cells, then, have to be loaded with 2.5 μM of the Ca2+ indicator dye Fura-2 AM in phenol red-free DMEM which contains 20 mM NaHCO3 (pH 7.4).
- Then, 30 min incubation at 37 °C is required.
- The phenol red-free DMEM should be then washed and incubated for 15 minutes in phenol red-free DMEM which contains 20 mM NaHCO3 (pH 7.4) in order to allow the hydrolysis of the ester group.
- The round glass coverslip with the cells should then be placed on the stage of a Nikon Eclipse TE200-E microscope.
- Add 300 μl of phenol red-free DMEM which contains 20 mM NaHCO3 (pH 7.4).
- Localize the cells to be recorded (as many as possible) and mark them as well as 2-3 background regions using the MetaFluor Software.
- After that, obtaining images of the loaded cells under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams is needed, and then the intensity of light emission at 505 nm every 5 sec needs to be measured.
- After 30-40 sec where the baseline is established, add the treatment (300 μl) and continue recording.
- Changes in [Ca2+]i after treatment has concluded are recorded with the use of MetaFluor Sofware. The background substrates ratios of the corresponding excitation wavelength (F340/F380) are also considered.
This protocol is adapted from Gahete, M. D., Luque, R. M. and Castaño, J. P. (2012). Measurement of Free Cytosolic Calcium Concentration ([Ca2+]i) in Single CHO-K1 Cells. Bio-protocol 2(22): e294. http://www.bio-protocol.org/e294
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