I have been performing a Annexin V/7-AAD timecourse of tumor cells transfected with different amounts of RNA. In one group getting a high dose of a virus derived RNA encoding GFP, I see the entire GFP positive population in the Annexin V-, 7-AAD+ quadrant. Are these cells necrotic? dead? late apoptotic?
I can start seeing the effect after about 24-48h, which is the time frame that the parent virus usually induces apoptosis. However, the amount of Annexin V+/7-AAD- cells is pretty low, suggesting either a very short apoptotic phase for CT26 cells or a non-apoptotic cell death.
Hi Erik,
Both apoptosis and necrosis are belong to programmed cell death. Apoptosis is further defined as early apoptosis (Annexin V+/7-AAD-) and late apoptosis (Annexin V+/7-AAD+). It is a continuous process from live, early apoptosis, late apoptosis to eventually necrosis or cell death, which is presented as Annexin V-/7-AAD+ .
The interpretation of this assay is attached below for your reference.
http://cyto.mednet.ucla.edu/Protocols/simultta.htm
Hi,
thank you for your input. I still don't know what the Annexin V-/7-AAD+ population is, as necrosis is described to be in the double positive population as well (also see here: https://www.researchgate.net/post/Whether_PI_Annexin_V-FITC_cells_are_classified_as_LateApoptotic_cells_or_Necrotic_cells1)
@ Tamara: I have observed the effect for Annexin-V PE & Annexin-V BV421.
Hi Tamara,
The RNA used to transfect GFP is cytotoxic itself too. Depending on the cell type and RNA type used, we observe a marked shift on the FSC/SSC Plot within 48-72h. We are looking into running this experiment with a Necrosis inhibitor to see if this population still exists and to validate the Annexin V staining too, maybe that will shed some light too.
As to the second part of your answer, do you suggest using 2 viability stains? What difference in staining are you expecting?
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