I am trying to study drug induced apoptosis by flow cytometry. Is it necessary to collect the soup of cell culture media while trypsisnising and pelleting the cells after desired treatments. since i see lot of floating cells and interested in apoptosis i do collect the cells in the soup for flow analysis. is the method correct?
its really not mandatory. the cells that floats are mostly dead , bole to apoptosis is already happen and now dead. if you do apoptosis for those attached cells you can still differentiate between early and late apoptosis between treated and control. so don't bother about the soups , you can directly trypsinize and proceed. those cells that float and dead will be removed during the gating process and will be towards bottom of SCC/FSC junction
I disagree with the previous two answers. You will dramatically underreport your apoptosis if you do not collect the supernatants. Yes, you will need to gate out debris with FSC & SSC, but you will want to include the apoptotic fraction.
Aliison is right. I was thinking of cell cycle analysis, sorry for the mistake. Apoptotic cells become more buoyant even though they are not completly dead.
All your dead cells will be apoptotic positive/ Annexin positive. But in reality its just fraction/minor thing if their is really a difference in apoptosis between the groups. If the cells are alive and apoptotic they will not be in the soup, and if they are dead because of cell cycle/or seeding and not of treatment. it will be same for both the groups. Just do both the conditions (collect just the soups label them with annexin) and (typsinize the cells and label them too) for both the groups and see the difference yourself , please repost to let me know your outcome.
thank you for answering the query. As of now i have collected the soup as i thought similar to allison. but i will post you when i repeat the expt.
To my view, Apoptosis study is incomplete if you do not compare and show the Necrotic/dead cell population as far as drug treatment experiments are concerned. For example, in Annexin V apoptosis kit, you will find 7-AAD which targets Necrotic cells. So, when you run it through flow cytometer, post the FSC/SSC plot you will do a quadrant gating to show the % of apoptotic, necrotic, dead and apoptotic+necrotic (in some cases this is important) cells. This will give you a complete data. Best of Luck.
I agree with Shyam that 7-AAD or PI needs to be included as well. I didn't notice before that it wasn't mentioned.
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