I am using the XTT Cell Viability Assay Kit (Biotium, Cat. No. 30007) to evaluate the cytotoxicity of a natural extract in human peripheral blood lymphocytes.

For those who have experience with XTT in PBMCs:

• What cell density per well (96-well plates) gives a reliable signal without saturation?
• What incubation time with activated XTT works best for lymphocytes?
• After 24 h treatment, do you wash the cells before adding XTT?
• Do you incubate XTT together with the culture medium or replace it with XTT solution only?

Any recommendations to maximize signal quality and minimize background would be greatly appreciated.