Matrigel coated glass bottom culture dishes

hESC (CHB-8 [27] ) cells were maintained on irradiated CF-1 MEFs (GlobalStem) in DMEM/F12 supplemented with 20% KnockOut Serum Replacement (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 13 GlutaMAX (Gibco), non-essential amino acids (Gibco), Beta-mercaptoethanol, and 10 ng/ml FGF2 (Sigma), or in feeder free conditions on Matrigel in mTeSR or E8 medium with 5× supplement (Stem Cell Technologies). For imaging experiments, cells were typically plated on matrigel-coated glass bottom culture dishes (MatTek). Differentiation of hESCs was induced with Activin A [28] (link) or retinoic acid. To label S phase cells, cultures were pulse labeled with EdU for 30 minutes.

Cells were seeded and cultured on Matrigel coated glass-bottom culture dishes (MatTek, 12- or 24-well dishes) for differentiation or treatments. Cultured cells were then washed with PBS and with BD Perm/wash buffer and then fixed with BD Cytofix at 4 °C for 15 min. Cells were then permeablized with 0.2 % TritonX-100 (in PBS) at room temperature for 30 min. Cells were then blocked with blocking buffer (2 % BSA and 1 % FBS in PBS) for 1 h at room temperature followed by staining with primary antibody diluted in blocking buffer at 4 °C overnight. The next day, cells were washed three times with blocking buffer before incubated with AlexaFluor secondary antibodies (Invitrogen, 1:1000 dilutions in blocking buffer) and DAPI for 1 h at room temperature. Cells were then washed three times with blocking buffer and mounted on glass slides (Vectors Labs) for imaging. All the primary antibodies used in this study can be found in Additional file 8: Table S7. Immunofluorescence images were collected using a Nikon A1R laser scanning confocal microscope with Plan Apo 10x, Plan Fluor 20x Ph1 DLL, or Plan Apo 20x DIC M objectives. Images were processed using NIS Elements or ImageJ. Some z-stacks were presented as maximum intensity projection images.