Hi Jawaher,

For cancer cell viability assays, I suggest using Alamar Blue instead of MTT. Alamar Blue is non-toxic, allowing repeated measurements on the same wells and providing more reliable results, especially when working with bacterial or natural product extracts. In contrast, MTT is an endpoint-only method and is prone to false positives/negatives because pigments or reducing metabolites can chemically reduce the dye, distorting the absorbance.

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Best,

Ahmad

Hello Jawaher Al Hamadi

The bacterial extracts may be causing metabolic inhibition and potentially detachment, which the MTT assay registers as reduced viability, leading to a reduced MTT signal but still have intact membranes (appearing viable in the Annexin V/FITC apoptosis assay). The Annexin V/FITC apoptosis assay does not measure the metabolic state of the cell. If your extract is inhibiting cell metabolism without triggering the specific cell death pathways (apoptosis, necrosis), this assay will report the cells as viable.

I suggest that

1. You test your bacterial extracts directly with the MTT reagent in cell-free media. If you get a purple color, it will indicate direct chemical interference.

2. Repeat the experiments with a more robust viability assay that doesn't rely on mitochondrial metabolism, such as the Alamar Blue assay as suggested by Ahmad Al-Khazaleh . The reduction is mediated by multiple enzymes in both the cytoplasm and mitochondria, reflecting the cell's overall reducing environment. While in MTT, the reduction is largely dependent on mitochondrial succinate dehydrogenase activity.

3. Annexin V/FITC specifically targets cells in early apoptosis, showing a positive stain where cells still have an intact cell membrane but have exposed phosphatidylserine (PS) on their outer surface. In this stage, Annexin V binds to the exposed PS, while cell membrane-impermeant dyes like propidium iodide (PI) cannot enter the cell. Therefore, early apoptotic cells appear Annexin V-FITC positive and propidium iodide-negative.

Best,

Dear Jawaher

Assume that at the IC50 dose, 50% of the cells have been killed and omitted by detachment and so on. In the flow cytometry-based PI/Annexin assay, you are practically performing the experiment on the remaining cells. Therefore, do not expect to see 50% dying cells here as well.

Maybe this attached paperon limitations of viability assays may help you. I would try a few other viability assays.