Goat anti rabbit alexa fluor 647 secondary antibody

Cells (1x10⁵) were seeded in 6-well dishes and cultured for 48 h. Cells were then washed twice with PBS and fixed with acetone for 10 min at room temperature. Next, the cells were blocked with 3% BSA-PBS (Beyotime Institute of Biotechnology) for 1 h at room temperature following permeabilization with 0.1% Triton X-100 for 5 min. Cells were then incubated with OCT2 (1:200) antibody for 2 h at room temperature, followed by Goat Anti-Rabbit (Alexa Fluor^® 647) secondary antibody (1:200; Abcam; cat. no. ab150079) incubation for 1 h at room temperature. DAPI was used to stain the cell nuclei at room temperature for 3 min. Fluorescent signals were detected using a fluorescence confocal microscope (Olympus Corporation; magnification, x400).

To prepare cells for flow cytometry, hiPSC-CF were dissociated using 1X TrypLE select (Thermo Fisher Scientific) for 10 min. Cells were centrifuged (200×g, 5 min, room temperature) and resuspended in cold 1% Paraformaldehyde for fixation (20 min, 4 °C). Cells were diluted 1:10 with 1X PBS and centrifuged (200×g, 5 min, room temperature). Cells were then rinsed once with Flow Buffer (1X DPBS, 1% Bovine Serum Albumin (Thermo Fisher Scientific) and 0.1% Triton X-100 (Sigma-Aldrich)) and stored at 4 °C. Fixed cells were then centrifuged (200×g, 5 min, room temperature) and resuspended in Flow Buffer with Rabbit anti-Vimentin primary antibody (1:200) (abcam). An unstained control group was incubated in only Flow Buffer. Cells were incubated with primary antibody or Flow Buffer overnight at 4 °C. Post-incubation, all groups were rinsed with Flow Buffer, centrifuged (200×g, 5 min, room temperature) and resuspended in Flow Buffer with Goat anti-Rabbit Alexa Fluor 647 secondary antibody (abcam) and incubated for 1 h at room temperature, protected from light. Stained cells were rinsed twice, and flow cytometry analysis was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec) and analysis performed using FlowJo (FlowJo, Ashland, OR, USA).