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You can use HBSS for mammalian cells...it's used all the time in the
Calcium assays on the FLIPR machines.  Just be sure to add HEPES (20 mM
final) to the 1X HBSS.

Regards,
Mary David
Molecular Devices
 ...now a part of MDS Analytical Technologies
www.moleculardevices.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Grzegorz Tylko
Sent: Wednesday, October 31, 2007 6:18 AM
To: [log in to unmask]
Subject: 10 mM CaCl2

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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
tel. +48 12 663 24 25
fax +48 12 634 49 51
e-mail: [log in to unmask]