Mouse anti oct4

Cells were washed twice with PBS, scraped with pre-heated Laemmli buffer (½ H₂O, ¼ SDS, ¼ Tris-HCl pH 6.8) and collected in an Eppendorf tube. Each lysate was boiled at 95 °C for 5 min and sonicated by passing the cells several times with through a 1-mL syringe. Protein concentrations was determined by BCA assay (Pierce, 23225). Proteins were separated by SDS-PAGE electrophoresis with a 4–12% Bis Tris gel (Thermo Scientific, NW0412C) and then transferred to a polyvinylidene difluoride membrane (BioRad, 162-0177). Blots were probed with mouse anti-Oct4 (1:1000, Cell Signalling Technology, 5677), mouse anti-Sox2 (1:10,000, Santa Cruz, sc365823) and rabbit anti-Actin (1:1000, Abcam, ab8227) antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1:10,000, BioRad, 170-6515 and 170-6516). Bands were detected following incubation with ECL (Pierce, 32106) by imaging on the iBright 1500 (Invitrogen) device.

Cells in 6 well plates were washed twice with PBS and treated with 0.25% trypsin-EDTA (Life Sciences) for 5 minutes to obtain single cell suspensions. Trypsin was inactivated with medium containing 10% FBS. Cells were counted, centrifuged at 300g for 5 min, washed twice with PBS and subsequently fixed in 2% paraformaldehyde (USB). Following 15 min incubation at room temperature cells were washed in PBS and in perm/wash buffer (BD) and resuspended at 4x10⁶ cells/ml in blocking buffer comprising perm/wash buffer with 0.1mg/ml human IgG (Sigma) and 10% serum from the species of secondary antibody (Life Technologies). Cells were incubated for 30min at 4°C and 50μl aliquots (2x10⁵ cells) transferred to individual 5 ml polystyrene round-bottom FACS assay tubes. For double staining of cells for OCT4 and SOX17, cells were incubated in perm/wash buffer with mouse anti-OCT4 (Cell Signaling) for 1h at room temperature, following by two washes with PBS and incubation for 1 hour at 4°C with goat anti-mouse-FITC (Molecular Probes) and goat anti-SOX17-APC (R&D Systems). Samples were washed twice and resuspended in 0.2% FBS in PBS in a final volume of 300μl/tube. Separate staining for OCT4 and SOX17 was performed analogously. Cells were analysed on a FACSCalibur flow cytometer (Becton Dickinson) and data analysed using CellQuest software.

Single-cell suspensions were fixed in ice-cold methanol (10 min at −20 °C) and permeabilized before incubation with primary antibody (mouse anti-OCT4 [Cell Signalling], goat anti-human SOX9 [Millipore]). Flow cytometry was conducted using a BD Biosciences Fortessa and the software Diva gating was used for analysis.