Fig. 1

Effects of medium changes on BrdU incorporation in GM25256 iBECs. a Representative immunocytochemistry of BrdU (green) incorporation in iBECs on days 2 and 9 after subculture, counterstained with DAPI (blue). b Schematic depicting days of iBEC fixation (X) or media change (MC) corresponding to graphs c–e. c Immunofluorescence analysis of BrdU + area/DAPI + area in iBECs with no MC on days 2 and 9 after subculture. Each data point represents the average of 3–4 random fields of view per well. Three independent differentiations were performed with n = 3 wells imaged per group per differentiation. ***p < 0.001 (Unpaired two-tailed t-test). d Immunofluorescence analysis of BrdU + area/DAPI + area in iBECs days 2 and 9 after subculture with no MC vs. on day 9 with a single MC on day 8. Each data point represents the average of 3–4 random fields of view per well. Two independent differentiations were performed with n = 3 wells imaged per group per differentiation. #p < 0.05, ***p < 0.001 (vs. day 2) (One-way ANOVA with Tukey’s multiple comparisons test). e Immunofluorescence analysis of BrdU + area/DAPI + area in iBECs on days 2, 4, 5, 7 and 9 after subculture with no MC vs. on days 5, 7, and 9 with continual MC on days 4, 6, and 8 (day 5: single MC on day 4; day 7: two MC on days 4 and 6; day 9: three MC on days 4, 6, and 8. Each data point represents the average of 3 random fields of view per well. Not significant (ns), ##p < 0.01, ###p < 0.001 (Two-way ANOVA with Tukey’s multiple comparisons test used to compare no media change vs. continual media change groups on days 5, 7, and 9). c–e Means are displayed with their SE