Cells are unevenly distributed across the membrane — poor reproducibility (Transwell Migration / Invasion Assay)

Question

Accepted Answer

Different fields of view show wildly different cell density; replicate inserts disagree. Common causes: 1) Cells were not mixed before seeding 2) Upper chamber not seeded evenly 3) Wells dried out unevenly 4) Operator handling varies between inserts Solutions: 1) Pipette suspension thoroughly before each insert 2) Add cells gently to the upper chamber and disperse evenly 3) Add medium immediately after seeding; avoid drying 4) Standardize timing and technique across all inserts