Isoton solution

Cells were harvested by trypsinisation, counted using the trypan blue exclusion method, and 1 × 10⁶ cells were transferred to 12 × 75 mm polycarbonate tubes. The cells were then washed twice in 2 mL flow cytometry buffer (0.5% (w/v) BSA in DPBS) by centrifugation at 400 g for three min at 4 °C. Cells were then fixed in 2% (w/v) paraformaldehyde for 10 min at 37 °C, washed, and then permeabilised with 1 mL 90% (v/v) ice-cold methanol on ice for 30 min. Following permeabilisation, the cells were washed and then incubated with a PE-conjugated murine anti-vimentin mAb (clone RV202, BD Biosciences) or the corresponding PE-conjugated mouse isotype control (IgG1κ-PE, BD Biosciences), in accordance with the manufacturer’s instructions. CD44 and CD24 staining was achieved by staining the cells without permeabilisation using FITC conjugated antiCD44 (eBioscience, clone 24E10) and APC conjugated CD24 (eBioscience, clone eBioSN3) antibodies. Prior to flow cytometric analysis, the cells were washed twice and then re-suspended in 300–400 μL of Isoton solution (Beckman Coulter) on ice. Cells were analysed using a Beckman Coulter Gallios flow cytometer. Histograms and dot plots were derived and analysed using Beckman Coulter Kaluza version 1 software.

Cell proliferation was undertaken using a Z2 particle size analyser (Beckman Coulter, UK) to count raw cell numbers; this was performed in both cell lines for comparison in triplicate. OE19 and OE33 cell lines were seeded into 24-well plates at a density of 2 X 10³ cells/well in 1 mL of RPMI 1640 medium. Cells were incubated for 24 h, and then the media was replaced with media containing the relevant treatments. Cells were then incubated for 72, 96, 120 and 144 h before counting; Each well was washed × 1 with 1 mL PBS and 0.5 mL of 1X trypsin added to each well, which was neutralised with 0.5 mL RPMI + 10% FBS once cells had detached. The well contents were transferred to coulter count cups containing 9 mL of isoton solution (Beckman Coulter, UK) and cells were counted using the Z2 particle analyser.