Recovered cells were trypsinized in 0.05% Trypsin-EDTA (Invitrogen) and resuspended at a concentration of 1610 7 cells in 100 ml PBS with 3% FBS. Two microliters of the rabbit anti-CCSP antibody (Millipore, Billerica, MA) was added, followed by a 30 min incubation on ice. Cells were washed twice in PBS with 3% FBS, then 2 mL goat anti-rabbit-FITC secondary antibody was added and incubated on ice for 30 min. After two washes in PBS with 3% FBS, cells were resuspended in the same but fresh media. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control. CCSP positive (CCSP + ) and negative (CCSP 2 ) fractions were obtained by fluorescence-activated cell sorting (FACS) using Vantage SEH cell sorter (BD, Bedford, MA). They were examined by immunfluorescence, adherent or 2D and sphere cell (3D) cultures and qRT-PCR.