Hi,
We've been trying to perform Ca2+ flux experiments by dying cells with FURA-2, and then measuring fluorescence using a plate reader in a 96-well format. The protocol has worked beautifully previously, but lately the cells detach every time during the staining step (we have tried with macrophages and chondrocytes).
Does anyone have any experience with this dye or have any suggestions as to what is going wrong? Everything we are using, the PBS, the FURA, the plates are the same batch as when the assay worked and everything has been kept sterile. At the end of the staining step (1 hour 37C), the cells detach when washed.
Thanks for any suggestions.
Hefin
Hi Helfin, are you performing the FURA-2 labelling in PBS? If so, I would in fact be surprised if the cells didn't detach, as they need cations such as calcium (which is absent from standard PBS) to maintain adhesion. Perhaps try labelling in HBSS instead?
hanks for your answer Lisa, we had considered it but the protocol is the same as we have used previously. Nevertheless it's a good suggestion which I'll try. Thank you!
I also think it is due to depletion of cations. Perhaps you had other cell culture consumable in older days where cells adhered better. You could think about spinning the plates for washing this would retain the cells
regards
Sven
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