Measurement of Free Cytosolic Calcium Concentration ([Ca2+]i) in Single CHO-K1 Cells

Materials and Reagents

1. Ca²⁺ indicator dye Fura-2-acetoxymethyl ester (Fura-2 AM) (Molecular Probes)
   
2. Phenol red-free DMEM
   
3. NaHCO₃
   

Equipment

1. 25 mm round glass coverslips
   
2. Nikon Eclipse TE200-E microscope with attached back thinned-CCD cooled digital camera (ORCAII BT; Hamamatsu Photonics)
   

Software

1. MetaFluor Software (Imaging Corp)

Procedure

1. Plate 50000 CHO-K1 cells/ml onto 25 mm round glass coverslips for 24 h.
   
2. Load the cells with 2.5 μM of the Ca²⁺ indicator dye Fura-2 AM (Molecular Probes) in phenol red-free DMEM containing 20 mM NaHCO₃ (pH 7.4).
   
3. Incubate 30 min at 37 °C.
   
4. Wash with phenol red-free DMEM and incubate in phenol red-free DMEM containing 20 mM NaHCO₃ (pH 7.4) for 15 min (to allow the hydrolysis of the ester group).
   
5. Mount the round glass coverslip with the cells on the stage of a Nikon Eclipse TE200-E microscope.
   
6. Add 300 μl of phenol red-free DMEM containing 20 mM NaHCO₃ (pH 7.4).
   
7. Localize the cells to be recorder (as many as possible) and mark them as well as 2-3 background regions using the MetaFluor Software.
   
8. Acquire images of the loaded cells under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and measuring the intensity of light emission at 505 nm every 5 sec.
   
9. After 30-40 sec where the baseline is stablished, add the treatment (300 μl) whose effect want to be proven, and continue recording.
   
10. Changes in [Ca²⁺]i after treatment administration are recorded as background substrates ratios of the corresponding excitation wavelength (F340/F380) using MetaFluor Software.
    

Acknowledgments

This protocol is adapted from Cordoba-Chacon et al. (2010); Cordoba-Chacon et al. (2011) and Duran-Prado et al. (2012).

References

1. Cordoba-Chacon, J., Gahete, M. D., Duran-Prado, M., Pozo-Salas, A. I., Malagon, M. M., Gracia-Navarro, F., Kineman, R. D., Luque, R. M. and Castano, J. P. (2010). Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents. Cell Mol Life Sci 67(7): 1147-1163.
   
2. Cordoba-Chacon, J., Gahete, M. D., Pozo-Salas, A. I., Martinez-Fuentes, A. J., de Lecea, L., Gracia-Navarro, F., Kineman, R. D., Castano, J. P. and Luque, R. M. (2011). Cortistatin is not a somatostatin analogue but stimulates prolactin release and inhibits GH and ACTH in a gender-dependent fashion: potential role of ghrelin. Endocrinology 152(12): 4800-4812.
   
3. Duran-Prado, M., Bucharles, C., Gonzalez, B. J., Vazquez-Martinez, R., Martinez-Fuentes, A. J., Garcia-Navarro, S., Rhodes, S. J., Vaudry, H., Malagon, M. M. and Castano, J. P. (2007). Porcine somatostatin receptor 2 displays typical pharmacological sst2 features but unique dynamics of homodimerization and internalization. Endocrinology 148(1): 411-421.
4. Duran-Prado, M., Gahete, M. D., Hergueta-Redondo, M., Martinez-Fuentes, A. J., Cordoba-Chacon, J., Palacios, J., Gracia-Navarro, F., Moreno-Bueno, G., Malagon, M. M., Luque, R. M. and Castano, J. P. (2012). The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells. Oncogene 31(16): 2049-2061.
   
5. Martinez-Fuentes, A. J., Moreno-Fernandez, J., Vazquez-Martinez, R., Duran-Prado, M., de la Riva, A., Tena-Sempere, M., Dieguez, C., Jimenez-Reina, L., Webb, S. M., Pumar, A., Leal-Cerro, A., Benito-Lopez, P., Malagon, M. M. and Castano, J. P. (2006). Ghrelin is produced by and directly activates corticotrope cells from adrenocorticotropin-secreting adenomas. J Clin Endocrinol Metab 91(6): 2225-2231.