Perm wash buffer

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.

Stimulation tubes were removed from the incubator in the morning to add monensin (0.7 µg/ml final concentration; BD Biosciences) and brefeldin A (1 µg/mL final concentration; Sigma-Aldrich; St. Louis, Missouri) and incubated for an additional 6 hours. Cells were then washed once with PBS and stained for viability with Aqua amine-reactive dye (Invitrogen) for 10 minutes in the dark at room temperature. A mixture of antibodies used for staining surface markers was added to the cells and kept at room temperature for 20 minutes. Cells were washed with PBS containing 1% bovine serum albumin (BSA, Fisher Scientific) and 0.1% sodium azide (Fisher Scientific) and permeabilized for an additional 20 minutes at room temperature using the Cytofix/Cytoperm kit (BD Pharmingen). Next, cells were washed in Perm/Wash buffer (BD Pharmingen), and a mixture of antibodies used for staining intracellular markers was added to the cells and incubated in the dark for 1 hour at room temperature. Cells were again washed with Perm/Wash buffer and fixed with PBS containing 1% paraformaldehyde (Sigma-Aldrich). Fixed cells were stored in the dark at 4°C until collection.