10 mM CaCl2

Posted by Grzegorz Tylko on
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Dear All,
we have just started fighting with calcium measurements in invertebrate cells (haemocytes) using Calcium Green and Oregon Green BAPTA. We have found that cell loading with the dyes was quite easy. They exhibit quite high calcium levels, especially when being in apoptosis. Now, we are going to calculate calcium concentration in cell cytoplasm. As a control to our experiment we use mammalian cells in culture, which are vivid in relation to haemocytes (physiological apoptosis). Paging through many protocols, we found that to obtain the maximum concentration in cell cytoplasm, people uses ionomycine followed by 10mM CaCl2 solution. It is not a problem to dissolve CaCl2 in HBSS, which we use for haemocytes but when CaCl2 is added to PBS (for mammalian cells) phosphates react with calcium giving us white, dense solution. We tried different PBS compositions with lower phosphate concentration as well as HEPES but all mixtures lead us to phosphate crystals.
Could anyone help us with it? Probably, the way out from that is easy but...
Grzegorz

dr Grzegorz Tylko
Department of Cytology and Histology
Institute of Zoology
Jagiellonian University
Ingardena 6, 30-060 Krakow
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