I am working with HeLa cells that have been treated with an organic compound. I use concentrations of organic compounds 3,6,12 ppm. Then I pass the flowsitometry with Annexin V-I which confuses me that the percentage of cells that experience apoptosis is very high at 3ppm at 43.5% while for 6ppm only 19.85% and 12ppm only 33.75%. is not the higher concentration of organic compounds apoptosis more and more cells? any one can help me resolve this problem?
Hi,
such results can indicate that the mode of action for toxic effects at the higher concentrations is not by apoptosis, but necrosis. Cells need time for the early apoptotic membrane changes detected by Annexin; if compounds act too fast/harsh, e.g. via direct membrane damage, you will not see early apoptotic cells (Annexin +, PI -), but only necrotic or late apoptotic ones (both PI +). Your cells should appear positively in the membrane integrity staining, I assume you did this in parallell?
On the other hand, the dose-response in your case is somewhat peculiar, so your results (n=1, I assume?) can also be caused by random experimental variation. Maybe run the assay one or two additional times and see if it gives more coherent results.
Programmed cell death often occurs by various combinations of modes and mechansms depending on both what and how much is used as an inducer. The speed of cell degeneration also affects the co-existance of signs of distinct mechanisms recruited by the inducer. I suggest you look into the current understanding of multiple cell death pathways and their known mechanisms, as well as critical reviews of the use of annexin V as a probe of apoptosis, for example in the following publications:
Exp Oncol. 2012 Oct;34(3):255-62.
Cytometry of apoptosis. Historical perspective and new advances.
Wlodkowic D1, Skommer J, Darzynkiewicz Z.
Cell Death Differ. 2018 Mar;25(3):486-541. doi: 10.1038/s41418-017-0012-4. Epub 2018 Jan 23. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Galluzzi L et al
patrick thanks for your answer, right i did this experiment in parallel. doses of the compounds I use do not use calculations based on IC50 of these organic compounds. maybe that's why the percentage value not so good, right? i used dysobinin compound with IC50 value is 5.94 micromolar
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