Hi, we culture human MSC in alpha-MEM+10%FBS and the basic media contains 0.8mM MgSO4, 1.8mM CaCl2, 5.5mM Glucose, and 117mM NaCl.
I want to prepare a solution containing 0.8 mM MgCl2, 1.8mM CaCl2, 5.5mM Glucose, 140 NaCl and 20mM HEPES to carry out the calcium imaging experiments. Does it make sense? Is the glucose essential?
What is the best for the calcium-free solution? I thought to use 5mM EGTA, and additional 1.8mM MgCl2 to compensate the content of divalent ions. Can I reduce the EGTA?
Do you think is needed to check the osmolarity?
Thank you!
During the experiments we perfuse our cells with a physiological buffer:pH 7.4, containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose and 1 mM HEPES.
For calcium free buffer we use the same composition and in addition 1mM EGTA, but without calcium.
Glucose is essential for such experiments to keep cells under normal physiological conditions.
Osmolarity is given by Glucose. If you want to avoid glucose, you can use equimolar amount of other sugars such as mannose.
Emrah, don´t you compensate the lack of CaCl2 with MgCl2 or NaCl?
Hi Ivan, no I do not, and so far (couple of years) it was not a problem to do so.
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