Human blocking reagent

Cells were collected with accutase (Innovative Cell Technologies, #AT104), washed twice with Assay Buffer: MACs rinse plus 0.05% BSA (Miltenyi, #130-091-222) and plated at 2x10^5 cells/well in a 96 well plate. Fc was blocked for 10 minutes in human blocking reagent (Miltenyi #120-000-442). Cells were pelleted and then resuspended in buffer containing either isotype (eBioscience #12-471-42) or DR5 (eBiosciences #12-9908-73) PE conjugated antibodies. Incubation occurred for 30 minutes on ice. Cells were then washed twice in assay buffer, resuspended in assay buffer containing 7AAD at 10ul/ml (eBiosciences #00-6993-50) and analyzed via FACS on a Canto (BD Biosciences). Single, live, 7AAD cells were gated. The resulting PE MFI was determined with isotype subtracted from DR5 signal. All cell lines could not be run at one time; therefore Colo205 was included in each run to determine consistency between runs and to be used as a reference point. All cell lines were analyzed two to three times. The MFI for each cell line was then categorized based on the Colo205 control. Cell lines with MFI within 30% of Colo205 were considered similar in expression (medium level). Greater than 30% were considered higher expression (high level), and less than 30% were considered lower expression (low level).

Cells were suspended in buffer (PBS containing 2% FBS and 5 mM EDTA), blocked with human blocking reagent (Miltenyi Biotec) for 10 min at 4 °C, and incubated with the appropriate antibodies (Miltenyi Biotec): CD10-FITC (130–124-215), CD184-PE (130–117-690), CD19-APC (130–113-642), CD133-PE (130–113-108), CD166-PE (130–118-349) and CD44-APC (130–113-331). After washing the cells twice with buffer, the cells were analyzed by a FACS Canto II cytometer (BD Biosciences) or sorted by a Fusion and Astrios cytometer (BD Biosciences).

For FACS analysis, 1x10⁶ cells were trypsinized and suspended in 125 µL of PBS containing 2% FBS and 5 mM EDTA. Cells were blocked with 12.5 µL of human blocking reagent (Miltenyi Biotec) for 10 min at 4 ºC. Then, cells were incubated with 5 µL of anti-CD44-FITC (Miltenyi Biotec #130-113-331) and 5 µL of anti-CD24-PE (Miltenyi Biotec #130-095-953) for 30 min at 4 ºC. After washing the cells twice with PBS-FBS-EDTA, they were suspended in 500 µL of the same buffer and analyzed by FACS with the FACS Canto II cytometer (BD Biosciences). Experiments were repeated a minimum of three times independently, in triplicate samples.