Assessing cell viability by neutral red staining

Cell viability was measured using the Neutral Red (NR) uptake test. For this, NR (Sigma-Aldrich) was dissolved at 0.01% in 1x PBS and subsequently sterile-filtered. 3 ml of this solution were added to each well and incubated for 1 h at 37 °C. The staining solution was removed and the cells were incubated at 37 °C for 2–3 h to allow the stain to diffuse through the agar and to be taken up by the viable cells. Pictures were taken at each millimetre of the scratch of each well. Per area the number of stained (viable) and unstained (dead) cells was counted and percentage of viable cells determined. Once viability was determined, cultures could not be used anymore for further migration and life/dead analysis evaluation.