Apoptosis by FACS - How to gate the cells by using Apoptosis kit from BD (Jan/01/2009 )
Hi:
I would like to detect the effects of one drug for my cells by using apoptosis kit from BD after incubation for 48 hours. My wonder is that I know my drug will kill my cells compared with untreated cells. So, the viabilty of cells from treated group and untreated group will different. By using the FACS machine, the first step I need to gate the cells from FSC and SSC dot plot. Which kind the of cells I need to gate in order to detect the annexin V-FITC and PI? The whole population or only the viable cells (without the cells debris and dead cells in FSC/SSC dot plot.
Thank you!
-feifeilike-
QUOTE (feifeilike @ Jan 1 2009, 11:46 PM)
Hi:
I would like to detect the effects of one drug for my cells by using apoptosis kit from BD after incubation for 48 hours. My wonder is that I know my drug will kill my cells compared with untreated cells. So, the viabilty of cells from treated group and untreated group will different. By using the FACS machine, the first step I need to gate the cells from FSC and SSC dot plot. Which kind the of cells I need to gate in order to detect the annexin V-FITC and PI? The whole population or only the viable cells (without the cells debris and dead cells in FSC/SSC dot plot.
Thank you!
I would like to detect the effects of one drug for my cells by using apoptosis kit from BD after incubation for 48 hours. My wonder is that I know my drug will kill my cells compared with untreated cells. So, the viabilty of cells from treated group and untreated group will different. By using the FACS machine, the first step I need to gate the cells from FSC and SSC dot plot. Which kind the of cells I need to gate in order to detect the annexin V-FITC and PI? The whole population or only the viable cells (without the cells debris and dead cells in FSC/SSC dot plot.
Thank you!
I will advise you to gate viable population first, although the number of viable cells is expected to decrease for drug treated cells.
either way, the viable healthy cells will be both negative, early apoptotic cells will be Annexin+ PI-, late apoptotic cells and dead cells will be both positive.
dead cells will be eventually fall into small fragments.
I hope this helps
-Rnotk-