Immunocytochemistry

Cells were cultured on a coverslip and fixed using 4% PFA. Fixed cells were washed several times in PBS and in PBT (PBS, 0.1% Triton) and blocked in PBT supplemented with 10% FBS. Primary antibodies were diluted in PBT 10% FBS at the following concentrations: anti-NANOG (0.5 μg/mL, Peprotech 500-P236), anti-LIN28 (2 μg/mL, ab63740), anti-OCT4 (2 μg/mL, ab19587), anti-SOX2 (2.5 μg/mL, R&D Systems MAB2018), anti-SSEA4 (4 μg/mL, ab16287), anti-CDH1 (1.25 μg/mL, BD610181), anti-SSEA1 (1/100, Hybridoma Bank MC-480) and revealed with secondary antibodies coupled with Alexa488 and Alexa 561. Nuclei were labeled with Vectashield DAPI mounting medium. Visualization and capture were realized with a Zeiss microscope and the Volocity software. Immunocytochemistry on differentiated embryoid bodies was performed using the 3-Germ Layer Immunocytochemistry kit (Thermo Scientific).

For FACS analysis, 10⁶ cells were suspended in 10 μl of PBS 1% BSA and 1 μl of PE-conjugated or V450-conjugated mouse anti-SSEA1 (clone MC480), rat anti-SSEA3 (clone MC631), mouse anti-SSEA4 (clone MC813) and mouse anti-TRA-1-60, all purchased from BD Biosciences. After 30 minutes incubation at room temperature, cells were washed in PBS and suspended in 200 μl of PBS and analysed by flow cytometry (MACSQuant, Miltenyi). For FACS analysis on differentiating embryoid bodies, dissociated cells were washed in 1xPBS, incubated with DAPI for 2 minutes for live/dead staining, fixed with 1% PFA-PBS for 20 mins at 4 °C and then permeabilized with 0.5% Triton X 100 – PBS for 20 mins at room temperature. Antibody staining was performed in 1% (w/v) BSA-PBS for 20 mins on ice, all antibodies were used in 1:20 dilution. The following antibodies were used: anti - Human/Mouse Brachyury PE (polyclonal goat IgG) R&D Systems and anti-Human Nestin FITC (Monoclonal Mouse IgG1 Clone #196908) R&D Systems. Compensation was performed by using OneComp eBeads (eBioscience).