Could you give a brief description of your experiment? Are you reading the plates with fluorescence or absorbance?

Hello Anjie Bispat

You could refer to the paper attached.

https://www.nature.com/articles/s41598-020-62848-5.pdf

The paper attached highlights the importance of assay optimization for each of the cell line to minimize the effect of confounders. They have pointed out that cell line, drug type, dose and treatment time have a substantial effect on cell viability. Also, seeding density, medium type and volume, and resazurin assay time were also shown to have an impact on cell viability, though to a lesser extent.

Some more highlights:

Pharmaceutical drugs are typically dissolved in various solvents (e.g., DMSO, DMF, saline, PBS) for use in in vitro studies. However, solvents such as DMSO can have a profound effect on cell viability, even at concentrations as low as 0.33%. By using a single DMSO vehicle control, we not only observed both over- and underestimation of viability, but also dose-response curves starting at levels above 100% viability.

In contrast, matched DMSO concentration controls are highly recommended as they reduce the risk of dose curves starting at >100% viability. Due to the risk of evaporation, matched controls and drugs should be plated in the same location on the 96-well plate. Subsequent use of matched concentration controls is more time consuming because e.g., a dilution series of the solvent needs to be prepared, which takes up more space on the 96-well plate and drives up costs.

In the edge effect experiments, we concluded that evaporation was higher around the perimeter of the 96-well plates, thereby affecting the concentration of added components and cell viability measurements. To minimize the edge effect, we excluded perimeter wells and instead filled these wells with PBS.

Since the cell viability assay is usually performed over several days, each 96-well should be seeded with an appropriate cell number so that the cell population will be in the exponential phase during the course of the experiment and not affected by high confluency (stationary phase) that in turn could affect drug efficiency.

Finally, an evaluation of evaporation on plates containing diluted drugs (stored at −20 °C) demonstrated that 96-well fat-bottom plates sealed with parafilm had higher evaporation than 96-well PCR plates sealed with aluminium tape. Consequently, evaporation of diluted drugs and medium components can lead to fluctuations in drug response, thereby making it difficult to estimate accurate IC50, GR50 and GRmax values and achieve reproducible results.

Best.

Could you read the plate with a fluorescence reader? It has been proposed to have better sensitivity than absorbance