Why most people use HBSS to do calcium assay?
I am now using HBSS to do this but I have to incubate the cell for more than 6h and I found that the cells were not good after such long incubation (compared to 2h incubation, the baseline 340/380 ratio increased and the cells did not respond to the stimuli anymore which can stimulate them at 2h incubation). So I want to know if I can use DMEM to do the assay or I heard from someone else that BSA in the buffer can help the cells to be stable for longer incubation, is that right?
Thank you for answer this question.
Please check the concentration of Calcium ions in both the media, the one with least Calcium ion should be used...so as to not interfere with actual results
Have you considered the temperature? Any buffer must have this composition: 20mM Hepes pH 7.4 (140mM NaCl, 5 mM KCl, 2mM MgCl2 and 1mM CaCl2, and 37`C for human cells. On other hand, the BSA and serum could interfere in the Fura loading.
Hi Francisco, I always keep my in the incubator (37C, 5% CO2) and in the FlexStation it is 37C. My buffer is a little different from yours. Mine is 140mM NaCl, 5mM KCl, 1mM MgSO4 and 2mM CaCl2, 10mM D-Glucose and 10mM HEPES, pH 7.4. BSA is not included in the loading buffer but included in the working buffer.
I know HEPES is used to buffer the solution without CO2, do you think I should not keep the cells in the incubator with 5% CO2?
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