I am currently working on measuring intracellular calcium levels when they are treated with Bapta-AM. I plated the cells at 40,000 cell/well/100 uL overnight in a 96 well plate and then loaded them with 100 uL of fura-2 loading dye. Do I treat the cells right before I monitor the fluorescence or do I do that beforehand? I think I am a little confused on the principle of the assay.
Load cells with Fura-2 AM for 20-30 min.Wash. Incubate further 20-30 min to allow full de-esterification of the Fura (the esterified form will not bind calcium as the AM is esterified onto the acidic groups that would bind calcium ions). Treat and assess fora fluorescence radiometrically.
BAPTA-AM will chelate intracellular calcium. What are you trying to measure? Fura-2 will remain in the cytosol for a lot longer if you load, wash and incubate at room temperature. If you maintain the cells at 37 degrees during loading the fura-2 will begin to enter the intracellular compartments such as the ER and you will have falsely high reads. This is especially important if you are measuring intracellular calcium release in response to specific stimuli.
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