Firstly, you must make sure that the annexin V/PI dual stianing kit does not deteriorate. Second, you should repeat the experiment two or more times under the guidance of specification or professional mentor. At last, you should exclude the false positive absorption of MTT due to the influence of the drug.

Once used a lower concentration of FBS to reduce the proliferation rate of cells over the weekend. The cells seemed to have not proliferated as they would normally do, maybe this is a simpler way you could try.

From what I understand, your extracts lead to reduced MTT readings (MTT is a viability assay. Reduced readings could mean increased cell death or reduced proliferation) but you don't see a difference when looking at Annexin/PI staining by Flow and you are wondering why.

It could be that your cells are not being killed but just stopped from proliferating or that your Annexin/ PI staining and Flow cytometry needs optimization. A few things you could do.

1. Stain your extract treated/untreated cells with PI/Hoechst and do Fluorescent microscopy. This will be an easier way of checking whether your cells are dead (PI+)

2. Treat your cells with your extracts +/- apotosis inhibitors (eg. zVAD) or necroptosis inhibitors (eg. Nec-1) and see if you see increased MTT values with one of the inhibitors. This will tell you whether your cells are actually dying when treated with your extracts and if so, which kind of cell death it is. You could follow this up with western blots for apoptosis/necroptosis markers to get some mechanistic insight.

3. If you would like to do Annexin/PI Flow staining, add a positive control where you should see death and make sure your technique is working properly. You can find plenty of protocols online.

Julie

Thanks Julie for the informative answer, maybe I will check this through AO/PI staining and check the viable and death cell to confirm the result of flow cytometry