Measurement of cell surface CD44 expression

HMM cells were plated on 6-well plates in triplicate. After 48 h of hypoxia incubation, HMM cells resuspended in PBS were incubated with primary antibody against CD44 (Genetex, CA, USA) at 4 °C for 1 h in the dark. Secondary antibody goat anti-rat IgG-PE (Santa Cruz, CA, USA) for CD44 analysis was incubated at room temperature for 30 min in the dark. After washing with PBS, HMM cells were resuspended in PBS and subjected to flow cytometric analysis for CD44 expression. Unstained cells were used to gate on live cells. After excluding cell debris from the gated populations, a minimum of 10,000 events per condition were collected for the analysis.