I'm using 7AAD together with TOPRO 3 and annexin 5 FITC.
My goal is to identify the cell death (or better apoptosis) and pannexin 1 channel opening by flow cytometry.
I know that Topro3 is getting in cells when they are permeabilized or when pannexin 1 channel is open.
I know that pannexin 1 is mainly activated during apoptosis as first signal.
I'm curious and I calculated the MFI of Topro3 in the Live cells when my cell where incubated with an inhibitor of Pannexin 1.
Although Topro3 MFI in live cells is less than in the apoptotic cells I observe that the cells treated with pannexin 1 inhibitor have a lower Topro3 uptake.
I checked out the unstained sample and it's not autofluorescence. I checked also the other single staining, but nothing strange.
How is that possible? Can it be an artifact?
Thanks in advance.