Apc brdu flow kit protocol

hESCs under thyroid differentiation protocol were incubated with 10 μL/ml of 1 mM BrdU in culture medium for three hours, at several time points. Then, treated cells and controls were washed once with PBS, collected and prepared for flow cytometry immunostaining as follows: Matrigel drops (at least 4 samples per time point) were first digested with HBSS solution containing 10 U/ml dispase II (Roche) and 125 U/ml collagenase type IV (Gibco, Thermo Fisher) for 30–60 min at 37 °C; then a single-cell suspension was obtained by dissociation with TripLE Express (Thermo Fisher) for 10–15 min incubation at 37 °C, the enzymes were inactivated by addition of differentiation medium. After centrifugation, samples were rinsed with PBS, fixed and stained following the APC BrdU Flow Kit protocol (BD Biosciences). In addition, PAX8 antibody was used to stain the thyroid cells. NKX2-1^GFP, PAX8 and BrdU incorporated cells (BrdU + ) data were obtained and processed using an LSR-Fortessa X-20 flow cytometer and FACSDiva software (BD Biosciences). Unstained cells and isotype controls were included in all experiments. In addition, the percentage of NKX2-1^GFP cells was used to estimate the thyroid generation efficiency of our protocol. Also, NKX2-1^GFP/PAX8 cells were used to evaluate the thyroid fate commitment. Gate strategies are exemplified in Supplementary Fig. 1h–i.

Cells with stable C/EBPβ knockdown were sorted and plated to 40 % confluence. Cells were also transfected with RUNX1t1 and analyzed for BrdU incorporation after 48 h. Briefly, cells were incubated with 1 M Bromo-deoxy-uridine for 20 min and then trypsinized and harvested in ice-cold PBS. Cells were then fixed, permeabilized, and stained with fluorescent anti-BrdU antibody according to the APC-BrdU flow kit protocol (BD Biosciences®). Dead cells were stained with 7-AAD and BrdU-positivity was then assessed by flow cytometry.