I am isolating human neutrophils for intracellular calcium estimation using FURA 2AM. I am able to get good number of resting cells but they are getting activated and form clumps only when incubated with FURA 2 AM (1uM) at 37 C for 45 min. Please help me with this problem.I follow the procedure given in the attached article.
The loading protocol in that paper looks fine, but they specifically note that the BSA they use in their HBSS is endotoxin-free. Is your BSA endotoxin-free?
Thank you Yaron. I use endotoxin-free Human serum albumin and not BSA. I have even tried with HBSS without albumin and also with endotoxin-free PBS.
How is your Fura-2 AM primary stock prepared? Concentration? Vehicle?
Primary stock is in DMSO (1mM) , from the stock I add 1ul per ml of cell suspension (final FURA concentration is 1uM)
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