Flow Cytometry Analysis of TRAIL Receptors

The expression of TRAIL receptors on surface membrane was assessed by flow cytometry using PE-conjugated antibodies (R&D Systems) as previously described [14 (link), 18 (link)]. Briefly, cells were grown at 70% - 80% confluence and harvested by incubation with Trypsin (0.25% w/vol)-EDTA (0.53 mM) at 37 °C for 3 min. Trypsinization prevents the formation of cell clumps after harvesting which facilitates the subsequent flow cytometry analysis. The resulting cells were immediately washed twice in PBS. Aliquots (1 X 10⁵cells) were incubated in 25 μL PBS containing 1% goat serum for 15 min at room temperature. Afterwards, cells were incubated with 10 μg/mL anti-DR4-PE or anti-DR5-PE (mouse IgG1-PE and IgG2b-PE as respective control) for 45 min at 4°C in the dark. Duplicate samples were incubated with the respective control IgG-PE (mouse IgG1-PE and IgG2b-PE) under the same conditions. Cells were then washed twice with PBS and resuspended in 0.5 mL PBS for final analysis. All cell lines were processed under the same conditions which ensure a direct comparison of the relative expression levels of DR4 and DR5 on surface membrane.