Hello Sirisha Devarakonda

You may incorporate these suggestions in your protocol. Maybe it will help to a certain extent to get consistent results.

1. First and foremost, you need to do a cell number titration. Choose a seeding density that becomes 80% confluent after 24 hours in a 96-well plate before you treat the cells with the candidate drug. Generally, I seed on an average 25000 cells in 100ul per well for cells having doubling time of about 24-30 hours. VCap cells is slow growing having a doubling time of approximately 52-55 hours. So, you will have to accordingly vary the seeding density bearing in mind that you have to achieve 80% confluency after 24 hours. Over confluent cells on the final day of the assay may lead to inconsistent results.

2. Never pipette growth medium or wash buffer directly onto the cells. Be gentle and add solution gently to the side of the well to avoid harming the cells.

3. For adherent cells, I would recommend round bottom 96-well plates instead of flat bottom plates. This is because if you are using multi-channel pipette, it will generate enough pressure when expelling liquid from the pipet to cause cell detachment when using flat bottom plates. If the plate reader cannot read round-bottom plates, you may transfer 200ul of the well content to a fresh 96-well flat bottom plate before recording the absorbance.

4. If you must shake the 96-well plate, you may do so by setting the shaking or rotating speed to very low speed to prevent cells from detaching.

5. Never allow cells to remain exposed to air for long at any time during the assay (while changing the medium or washing the cells).

Other factors that may cause variation include the amount of reagent that actually enters the cell, cellular metabolic activity (which is highly dependent on a multitude of variables including treatment to the cells), the timing of formazan crystals extrusion, the cytotoxic effect of MTT, abiotic reduction of MTT by culture media or the tested treatment, MTT concentration and MTT incubation time, and finally the optical measurement which may also experience interference from the culture media or the tested treatment.

In such cases, complementary assays are recommended to assist in the interpretation of MTT assay measurements.

You may want to refer to the below attached paper for more information.

Article The MTT Assay: Utility, Limitations, Pitfalls, and Interpret...

Best.

Thank you Malcolm Nobre for the detailed information. I will adjust the cell number according to your suggestion.