Primary igg antibody against ews

Adherent Ewing’s sarcoma cells were detached from culture flasks by incubating with 0.02% EDTA (Sigma Chemical, St. Louis, MO) in PBS and then dissociated into monodispersed cells. Cells (1 × 10⁵) were collected and suspended in 100 μl of 1× flow cytometry buffer (1% BSA and 0.1% NaN₃ in PBS, filtered through the 0.22 μm filter unit) containing an FcBlocker (eBioscience, San Diego, CA) and incubated at 4°C for 15 min. Primary IgG antibody against EWS (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted (1:200) in 1% BSA in PBS and then added to the cell suspension. Parallel staining was also performed with rabbit IgG (1:200) (Jackson Immunoresearch, West Grove, PA) as an isotype matched control for EWS. After incubation at 4°C for 30 min, cells were washed twice with the 1× flow cytometry buffer. Cells were then incubated in 100 μl of 1× flow cytometry buffer containing FITC-conjugated goat anti-rabbit secondary IgG antibody (1:200) at 4°C for 30 min. Subsequently, cells were washed as above, fixed in 2% paraformaldehyde in PBS and then transferred into a 5-ml polystyrene round-bottom tube capped with a cell-strainer 183 cap (BD Biosciences, Franklin Lakes, NJ). Antibody-bound cells were then analyzed on Epics XL-MCL Flow Cytometer (Beckman Coulter, Fullerton, CA) using CellQuest software (BD Biosciences, Bedford, MA) and also FlowJo software (Tree Star, Ashland, OR).