Measuring Single CHO-KI Cells With Free Cytosolic Calcium Concentration

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
  • NaHCO3
  • Phenol red-free DMEM

Equipment and Software

  • Nikon Eclipse TE200-E microscope (or equivalent)
  • MetaFluor Software (or equivalent)

Method

  1. Plate 50000 CHO-K1 cells/ml onto 25 mm round glass coverslips for 24 h.
  2. 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).
  3. Then, 30 min incubation at 37 °C is required.
  4. 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.
  5. The round glass coverslip with the cells should then be placed on the stage of a Nikon Eclipse TE200-E microscope.
  6. Add 300 μl of phenol red-free DMEM which contains 20 mM NaHCO3 (pH 7.4).
  7. Localize the cells to be recorded (as many as possible) and mark them as well as 2-3 background regions using the MetaFluor Software.
  8. 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.
  9. After 30-40 sec where the baseline is established, add the treatment (300 μl) and continue recording.
  10. 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.

Acknowledgements

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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Last updated: Jun 13, 2019 at 7:54 AM

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