Lsr2 fluorescence activated cell sorter

Cell mono-layers were washed with PBS and detached with Versene (supplied as a 1× solution of 0.2 g/l ethylenediaminetetraacetic acid, Life Technologies, Carlsbad, CA, USA) at 37°C. Following centrifugation at 500×g, the cells were washed in PBS and re-suspended in FACS buffer (PBS, 0.5% w/v BSA, 0.1% NaN3). The Fc receptors were blocked by the incubation of 5 × 10⁵ cells per tube with 1 µg human IgG at 4°C for 30 min. For the detection and quantification of TRAIL R1 and R2, 2.5 × 10⁵ cells were incubated with antibodies either, TRAIL R1 (PE conjugated, FAB347P, 25 µg/ml) or R2 (Alexa 488 conjugated, FAB6311G, 50 µg/ml), the appropriate IgG control, mouse IgG₁- PE conjugated (IC002P, 25 µg/ml) or mouse IgG_2B – Alexa 488 conjugated (IC0041G, 50 µg/ml), respectively as per the manufacturer's instruction. Compensation beads were used to reduce background interference (BD Bioscience, Oxford, UK). Data were collected on an LSR2 fluorescence activated cell sorter (BD Biosciences) and analysed using the Flowjo software package (Ashland, USA). Antibodies were all purchased from R&D systems (Minneapolis, MN, USA).