I would like to count the macropinocytosis levels on MiaPaca-2 but I am a bit confused about the incubation time with the EIPA inhibitor and the Fitch-dextran. After I sees the cells and let them adhere, I starve them overnight and on the next day I treat them with EIPA diluted in serum free media for 30min at 37oC. After the incubation time passes, I aspirate the media and I add fresh serum free media with fitch-dextran 1mg/ml for a total of 30min at 37oC. Then I wash and I fix with 4% PFA and I image.
Even though I have sufficient inhibition effect by the EIPA,I don’t know if i should better pretreat the cells with EIPA diluted in serum free media for 30min at 37oC and then add on top of it 1mg/ml of fitch-dextran for an additional half an hour at 37oC.
Does anyone know ?
Thank you very much!
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