How to maintain cells at a specific apoptosis stage - (Mar/07/2009 )
I want to test a compound for apoptosis induction. I need to take my cells in several times for annexin V assay.
But the problem is that I can only use flow cytometry on 12 o clock. Thus I cannot take my cells with 12 hours exposure to compound!!
there is the same problem with 6, 9 and 15 hours.
Can I centrifuge cell after for example 6 hours exposure to the compound and maintain them on the pure media for one day? are they going to be at the same apoptosis stage after one day? I myself doubt it.
Any idea would be certainly useful
thanks so much in advance
I knew methods for arrest of cells at G0 or s phase...but never heard of early stage or late stage arrest of apoptosis.....you need to search more.
Do you have to do annexin V assay?
Why don't you change methods to ELISA?
Apoptosis is an active cascade of signalling and events that I do not believe can be held in a specific stage. It will either move forward to cell death or reverse and attempt rescue/survival. Either way the intracellular conditions will greatly change with time. If you are a graduate student, it is time for the un-escapable overnighter! We all have had to do it at some point. I've slept on the couch in the breakroom to the glow of my timers. It's fun when the student in the lab next to me has to stay as well. Sort of a sleepover in the lab. You need to time each sample's treatment so they will be ready to harvest at the same time and able to be sorted at your alloted time. If you are a post-doc, you may be able to bribe a student to stay overnight and treat the cells for you.
unfortunately I have to do annexin V assay!
I need however to have the 9 12 and 15 hours exposure to the drug and I donot have any idea of how to do it
any idea about freezing cells in -70 degree to maintain cells in that specific stage of apoptosis for one day?
Once cells have started apoptosing, there is very little that can be done to prevent them dying. Freezing will not work as they will not survive the thawing process. However, the standard proceedure for FACS, at least for PI is to fix the cells in ethanol, and they will keep almost indefinitely after they are fixed. I don't know if this will work with annexin V as I have never done it, but you could try.
What you may need to do is to set up your experiments so that you harvest them all at the same time: This will mean that you will need to treat you cells at different times (i.e. 9 pm day before (15 hours before), midnight (12 hours before), etc).
Stain your cells with your Annexin antibody and wash appropriately and then fix them in formaldehyde (anywhere from 1 to 4%). You can keep them at 4 degrees until your run. You may want to play with the formaledhyde concentration to keep the strength of your signal. If you fix before you stain, you will get massive false positive. What i am not certain about is the counterstain (ie. propidium or 7-AAD), but at least you can keep your annexin V signal.
dear NemomeN and others,
thanks alot for your help.
just some questions. How much formaldehyde should I use? is it ok to add 900 microliter formaldehyde to 100microliter cells,annexin and Binding buffer, store them in microtubes at 4 degrees and run the next day?
what if I centrifuge cells after being fixed to wash away the formaldehyde and resuspend them in PBS? will I then have good strength of signal?
dear rkay447, I donot have any problem to become an overnighter!!!as you said it is fun. But the problem is that I am not allowed to stay in lab no later than 8 pm!!!!! and the flow cytometer technician never turn the flow cytometer on twice daily!!!
it is really problematic to work in molecular biology field in my country!!!!!!!!!!!
just one more question. should I stain with DAPI or annexin for flourescence microscopy before I fix as u said for flow cytometry?