OD values are too high — beyond detection range (CCK-8 Cell Viability Assay)

Question

Accepted Answer

OD450 readings approach or exceed 3.0, the curve hits its upper limit, and treatment vs control look indistinguishable. Common causes: 1) Seeding density was too high 2) Culture period was too long 3) Cells proliferate too fast 4) Wells reached over-confluence 5) CCK-8 reagent was overdosed or incubated too long Solutions: 1) Reduce seeding density (halve it, or run a density gradient) 2) Shorten the culture window (use multiple time points instead of one late one) 3) Aim for OD450 in the 0.1 – 2.0 range 4) Reduce CCK-8 reagent volume or incubate 1 – 2 h only 5) Keep well confluence under 80 %