The transwell migration assay is commonly used for assessing cell migration. It involves the enumeration of cells that
have migrated across a pore-containing membrane. We describe a randomised approach to quantifying migrated
cells and compare it to a conventional full cell count. We used ATP as a chemoattractant and automatic cell quantification performed on all fields (Full count; FC) or 10 randomly selected fields (Randomised count; RC). The two
methods were compared by evaluating standard deviations (SD), coefficient of variation (CV) and using the Bland-Altman analysis. The dispersion of data is higher with the RC approach (3.77-6.66% CV for control; 3.89-4.48% CV
for ATP-treated wells) compared to FC (0.27-0.46% CV for control; 0.05-0.09% CV for ATP-treated wells), but are
acceptable considering that the number of migrated cells are in the thousands. Both methods verified that an ATP
migration assay for BV2 microglia was established, demonstrating that the RC approach is reliable and comparable
to a full count.
Our work cautions against the use of serum-supplemented culture media in a transwell migration assay when using chemoattractants other than FBS. At 24 h, a 5% foetal bovine serum (FBS) gradient caused BV2 microglia to migrate toward the lower compartment of the transwell apparatus. Interestingly, FBS-supplemented media in the absence of a gradient also resulted in notable microglia migration. Serum can therefore confound the interpretation of a transwell migration assay when another chemoattractant is used.