Poor reproducibility — imaging position is inconsistent (Wound Healing Assay)

Question

Accepted Answer

Wells differ wildly between replicates; quantification jumps around with no clear pattern. Common causes: 1) Scratch position and angle differ between wells 2) Imaging field is not fixed 3) Seeding density and treatment timing differ Solutions: 1) Mark fiducial points on the back of the plate so you find the same field every time 2) Always image the same position 3) Standardize seeding amount, scratch time, and imaging time across the entire experiment