Some leukemic cells collected from patients undergo apoptosis when treated with a drug. Immunoblotting shows complete PARP cleavage.On the other hand, when I did Annexin V/ PI staining on the same cells after drug treatment, they did not exhibit any change from untreated cells in terms of annexin staining. The annexin staining, however, worked fine with other cell lines, with proper positive and negative controls.
Is this due to a problem with annexin staining or is it because of 'primary' nature of cells, being directly from patients? Do primary cell cultures (especially, leukemic cells) do not take up annexin stain? Do the cells need to be incubated with annexin for a longer time? Any help in this regard will be highly appreciated.
I suggest you consider evaluating your cells after 48h of exposure and do a dose response study. Primary cells will stain in the annexin fitc staining without any problems. www.ncbi.nlm.nih.gov/pubmed/17061978. Sometimes there are normal cells in the primary samples and if for some reason your FACS operator is gating on those cells, you may not see the annexin staining. Double check if your cells are showing shrinkage (which they should) on the light scatter plots. Good luck !
http://www.ncbi.nlm.nih.gov/pubmed/23568490
I suggest you consider evaluating your cells after 48h of exposure and do a dose response study. Primary cells will stain in the annexin fitc staining without any problems. www.ncbi.nlm.nih.gov/pubmed/17061978. Sometimes there are normal cells in the primary samples and if for some reason your FACS operator is gating on those cells, you may not see the annexin staining. Double check if your cells are showing shrinkage (which they should) on the light scatter plots. Good luck !
http://www.ncbi.nlm.nih.gov/pubmed/23568490
And also ask the FACS operator to give you a printout of the total population and not the cells that were gated on. if your cells all die and shrink, yhey will fall out of the gated window and you will no longer detect annexin stsining even though all of them may be annexin positive
I think it might be a timing problem, from my experience Annexin V staining is a late event in apoptosis whereas PARP clevage is early on. Also do you know that your cells are finally dying? Cells have mechanisms to evade apoptosis (Bcl-2, Bcl-XL) so you might detect early signals but the cells never (or only after a lag phase) go into apoptosis. You might want to check effector caspases.
Best!
Patrik
I agree with the above advice.. I work primarily with primary lymphoma samples. I suggest you set up the experiment where you examine treated and untreated cells over time with trypan blue stain and AXV. This will give you cell viability which you can compare to your ANV flow analysis. I do this as a confirmation that I'm gating on the correct cell population. Also as suggested above follow ANV over time. In my lab we follow ANV staining at 24, 48 and 72 hrs post treatment. You may also want to lower your drug concentration as you may be killing the cells too rapidly.
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