I worked with one drug that is cytototoxic on k562 cells upto 50 micomolar (98 % approx inhibtion. i did annexin v/pi flow cytometry assay but after 48 hours of treatment with different concentrations, i am getting only increase in pi+ cells and no fitc cells at all almost. however with postive control standard curcumin, i am getting both fitc + and Pi + cells varying with concentrations used. Should i conclude that the particular drug induces necrosis only and no apoptosis ? will there be possibly any effect if i decrease time point after treatment and i may get Fitc + cells?
I suspect that your drug of interest is allowing a small molecule across the membrane (PI), but not a large protein (FITC-Annexin V). This does not necessarily indicate death at all, but may be due to non-selective (but small) channel formation. If this was necrosis, you should definitely see PI+ and FITC+ because of loss of membrane integrity to small and large probes.
I would recommend you use a second method to test cell death. Alternatives: caspase 3/7 activity by fluorescence activity assay; PARP or casp-3 cleavage by western; DNA cleavage by TUNEL; nuclear fragmentation by DAPI staining. A clonogenic assay is an inexpensive way to assess death, but can be confounded by severe inhibition of priliferation.
hi Yaseen,
May I ask some question about your sample? Have you ever try to reduce the concentration your treatment? how about your range concentration? how about the results when you check the inhibition/cytotoxic assay, e.g. IC50 of your sample?
-dew
Dear MOHD YASIN BHAT Yaseen,
An increase in PI staining means that plasma membrane lost its integrity. It indicates either necrotic cells or old apoptocic cells. So you need to do further analysis to solve this issue. You can do the same procedure during shorter observation periods : 24 and 36h and/or you can measure cell cycle analysis to see whether there are hypodiploid = apoptotic cells.
Good luck!
P.M:
thank you Justin sir for your suggestion but i have one doubt that, why the result came good with postive control curcumin with increasing concentration including increase in both pi + and fitc+ cells. Also i have tested cell death with other assays previosly like mtt and there is dose dependent increase in death of cells
Well since you have done a dose response experiment in a 48h incubation period, you certainly have evidence of no apoptosis in your treatments. Never the less, have you done a time course experiment, before you make your final decision. Seems to me that 48 h is a very long time. Why don't you make 12 and 24h incubation periods. Although you probably should have seen already some PI+fluorescence positive cells, but you haven't, that almost convince me of your result.
I would do time course to make sure of my result.
And the other side of your question is, whether there are drugs inducing neat necrosis without signs of apoptosis, the answer is yes, there are anti-tumor drugs doing just that. Nice talking to you, bye.
Thank you Paula. and Petr. i am planning a time course experiment before concluding result. thanks for suggestion
The finding that the positive control worked is very important here. This means that if phosphatidyl serine was externalized, as happens in apoptosis, you should have observed FITC+ cells. This is part of the reason I am skeptical you are observing apoptotic death. Remember, these assays are not fool-proof. Positive staining is not the same as apoptosis and negative staining is not the same as healthy cells. Even though your positive control shows apoptosis, it doesn't mean that a change in signal caused by a different compound is also from apoptosis.
The MTT assay does not measure cell death. It measures, indirectly, the number of cells adherent to your plate. This assay is useful because it provides a rapid, quantifiable signal for low cost. But a reduction in MTT signal can also indicate mitochondrial toxicity (MTT needs to be metabolized), it can indicate impaired proliferation, or it can indicate loss of adhesion to the plate (cells wash away).
Finally, a concentration curve helps you determine the optimal and minimal concentrations that have an effect, but will not tell you why the signal (PI+ or FITC+ or MTT absorbance) is changing. I agree with looking earlier after treatment. If you still find that FITC-AnnxV remains negative, consider that your cells may not be apoptotic. You will need additional assays to convince yourself.
thank you Justin sir for sharing your expertise and giving suggestions. i will do some more experiments on this before concluding anything. May let you people know about the outcomes . Thanks a lot
apoptosis and necrosis should be analyzed using/testing agents at different doses and at different time points
Necrosis should be easily confirmed by release of Lactic dehydrogenase LDH in the medium. It is an enzymatic assay available in kits with all you need but a spectrophotometer. You may cause lysis of all cells adding some detergent to a control and give this amount 100% then the Sup of the negative control would be base level.
annexin V- FITC /propidium iodide assay is commonly used test for analysis of the frequency of early apoptotic cells and also late apoptotic and necrotic cells
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