[Cell-biology] annexin/ h2dcfda

Koschutnig Karin via cellbiol%40net.bio.net (by karin.koschutnig from univie.ac.at)
Thu May 24 14:45:46 EST 2007


I have just started to work with flow cytometry. 
I have tried h2dcfda to measure ROS in my cells (Hepg2). The problem is that my untreated cell are already 70-80% positive. 
What did I wrong? I use 5yM and incubate for 30 min. 

Then there is another thing I 'm not sure about. 
I have planed to measure apoptosis( annexin /7AAD) and H2DCFDA. I incubate my cell for 24h with different concentrations of my sample, which is quite cytotoxic. So when I seed for example 1 Mio cells, there will be different amounts of cells left. Should I resuspend these different amounts in 1ml of binding buffer and dye them. Or should I take 1 Mio for each concentration. As I have to count them by the trypan exclusion assay, this takes me quite long. I'm not sure is this time stress my cells even more. 

It would be nice if somebody could help me. 
best regards, Karin Koschutnig

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