Low background values in MTT assay?

Cell Metabolism ........Different cell lines may have variations in their metabolic activity, which can affect the reduction of MTT and subsequently influence background values. It's important to ensure that the cell lines are maintained under optimal conditions, including appropriate culture medium, passage number, and confluency, to minimize variations in metabolic activity.

Serum and Media Components ........The composition of the culture medium, particularly the serum and other additives, can impact the background values in the MTT assay. Try to use the same batch of serum and media components for all cell lines to reduce variability. Additionally, consider testing different serum concentrations or using serum-free media to determine if that affects the background values.

Cell Confluence and Seeding Density........The confluence and seeding density of cells can influence metabolic activity and subsequently affect the background values. Ensure that cells are seeded at appropriate densities and reach a consistent confluence across all cell lines tested.

Incubation Time: The incubation time with the MTT reagent can also affect the background values. Ensure that all cell lines are incubated for the same duration and follow the recommended incubation time for the MTT assay.

Reagent Preparation and Handling........Proper preparation and handling of the MTT reagent are crucial to obtain reliable results. Ensure that the MTT reagent is freshly prepared and stored correctly according to the manufacturer's instructions. Additionally, make sure the MTT solution is added consistently to all wells and handled in a sterile manner.

Troubleshooting and Controls........ Validate the assay by including appropriate controls. Positive controls (known cytotoxic compounds) and negative controls (untreated cells or cells treated with vehicle only) can help verify the sensitivity and specificity of the assay. If possible, compare the results of the problematic cell lines with other established assays, such as a trypan blue exclusion assay or ATP-based viability assays, to confirm the observation

Istvan Zupko

Dear George,

Thanks a lot for your points. Since the only detail we changed is the serum supplier, we will try to compare several different serums.

Thanks again.

Alireza Mohebbi

Hello Istvan Zupko

There it is normal to have background noises in MTT assay. If your instrument have the option to get ODs at two different weave lengths (570 and 630), the machine will reduce the noises due to adsorbed lights at higher weave length. If the noise is also observed in control cells along with test wells, you can standardize the ODs with the control.

Istvan Zupko

Dear Colleague,

Thanks for your suggestion. Two wavelengths are possible, but I am afraid that is not enough. Our point is the decrease of the metabolic activity of some cancer cell lines, and we can not identify the reason. We decided to test fetal bovine sera from different suppliers. Hopefully it will be conclusive.

kind regards

Istvan

Istvan Zupko

Dear Guarav,

thanks for your suggestion, we have tested for mycoplasma but additional test will be performed. Hopefully we will clarify the reason of our problem soon.

Thanks again

kind regards

Istvan

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