Fig. 1: NAD(P)H and FAD fluorescence change differently and reflect different glycolytic activity in the first 24 h for cells in low vs. high cardiomyocyte differentiation efficiency conditions.
From: Label-free imaging for quality control of cardiomyocyte differentiation
hPSCs were differentiated into CMs following an established method11. On differentiation day 12, cells were verified by flow cytometry with cTnT labeling from three independent replicates. a, b Representative flow cytometry dot plots for a low and b high differentiation efficiencies along with negative controls. Gating strategy to determine the percentage of cTnT positive population in hPSC-derived cells. Single-cell quantitative analysis of mean lifetimes (τm, reported as picoseconds) of c–e FAD and f–h NAD(P)H, and i–k optical redox ratio for low (0.3% cTnT+) and high differentiation (65.5% cTnT+) efficiencies on day 0 (“D0”, immediately pre-treatment) and day 1 (“D1”, 24 h post-treatment with CHIR99021), and their corresponding representative images. n = 2458, 633, 3534, and 4446 cells for 0.3% day 0, 0.3% day 1, 65.5% day 0, and 65.5% day 1, respectively. Data are presented as dot plots with bars for the mean and 95% CI for each condition each day. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests. ****p < 0.0001. Color bars are indicated on the right. Changes of optical redox ratio after treatment with 2DG or rotenone. l Single-cell quantitative analysis of optical redox ratio for H9 ESCs before and 2 h after 10 mM 2DG treatment. n = 1051 and 900 cells for before and after 2DG treatment, respectively. m Single-cell quantitative analysis of optical redox ratio for H9 ESCs before and 15 min after 10 μM rotenone treatment. n = 1042 and 986 cells for before and after rotenone treatment, respectively. Data are presented as dot plots with bars for the mean and 95% CI. Statistical significance was determined by unpaired two-tailed Student’s T-test. ****p < 0.0001. ps, picoseconds. After the first 24 h of differentiation, n lactate and o glucose concentrations of cell culture medium from low (10.8%) and high (63.1%) differentiation efficiency conditions were measured with three biological replicates, respectively. Data are presented as dot plots with mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s T-test. *p = 0.0210 and 0.0291 for n lactate assay and o glucose assay, respectively. Source data are provided as a source data file.
