How would you investigate a phenotype that disappears after cell passaging?

Why

First confirm when the phenotype disappeared and whether the assay protocol, reagent lots, incubation conditions or operator changed. Track the passage number, population doublings, confluence at seeding and growth rate, and test mycoplasma. Authenticate identity using an appropriate species-specific method, such as STR profiling for human lines.

Recover an early frozen stock and compare matched low- and high-passage cultures in a blinded experiment with the same controls. Long-term propagation can select subclones, alter gene regulation or change differentiation, but a single passage-number correlation does not prove genetic drift. Measure baseline markers and expression of the target pathway to distinguish changes in cell state from assay failure.

Document an acceptable working passage window and refresh seed stocks when the evidence supports it. Avoid repeatedly subculturing the failing population while troubleshooting because that can introduce more drift.