Mab2188

1.0 × 10⁶ cells were trypsinized and re-suspended in FACS Buffer (PBS pH 7.4+3% FBS). Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG_2a (R&D Systems, MAB003) at 1:100 for 30 min on ice. Cells were washed with 1 ml FACS buffer and pelleted at 1200 rpm for 5 min. The supernatant was aspirated and cells were incubated with anti-mouse IgG_2A AlexaFluor 647 (Invitrogen Life Technologies, A21241) at 1:200 for 30 min on ice. In the case of MDA231 cells growing in hypoxia, the above two antibody incubation steps were performed in the hypoxia chamber. Cells were washed and resuspended in 200 μl of FACS buffer containing 1:10 000 propidium iodide (Sigma-Aldrich, P4170). 1.0 × 10⁵ events were collected for each sample on a BD FACS Calibur (BD Biosciences, San Jose, CA, USA) and the data were analyzed using FlowJo Software (FlowJo LLC, Ashland, OR, USA).