Real-time monitoring of cell proliferation

Cells were seeded at 5000 cells per well in 200 μl of complete media in E-plates (ACEA Biosciences, San Diego, CA, USA) and grown overnight while monitored with an xCELLigence DP system (ACEA Biosciences) which monitors cellular events in real time by measuring electrical impedance across interdigitated gold micro-electrodes integrated on the bottom of tissue culture plates [16 (link), 17 ]. Cells were washed three times with PBS and cultured with 180 μl EGM-2 basal media (no growth factors or supplements) and incubated for a minimum of 6 h before further treatment. Treatments were prepared at 10 × concentrations and added to each well in a total volume of 20 μl. The xCELLigence DP recorded cell index readings every 15 min for 3 days after treatment. Cell index readings were normalized before treatment and cell proliferation ratios were determined from four biological replicates and represent the relative numbers of cells compared to control cells. A two-way ANOVA with Holm–Sidak’s multiple comparisons test was used to compare IPSE treatment to medium-alone control, with P ≤ 0.05 deemed significant.