Cd44 pacific blue

Primary Apc⁻; Pten⁻; Trp53⁻ tumors were mechanically dissected into single cell suspension as previously described (5 (link)). Cells from primary tumor suspensions or the W2476T cell lines were then isolated using fluorescence activated cell sorting (FACS). Briefly, primary ovarian tumor or W2476T cell line single cell suspensions were counted and incubated with primary antibodies CD24-PerCP Cy5.5, CD117-APC and CD133-PE (eBioscience, San Diego, CA), CD44-Pacific Blue (Biolegend, San Diego, CA), CD90-PE (BD Pharmingen, San Jose, CA) for 30 min at 4° C. Cells were then stained with propidium iodide (PI) or DAPI as a viability stain. For ALDH⁺ samples, ALDH enzymatic activity was defined using the ALDEFLUOR kit (Stem Cell Technologies, Canada) as previously described (5 (link)). FACS was performed with ~ 1 ×10⁶ cells using FACSAria (Becton Dickinson, Franklin Lakes, NJ) under low pressure in the absence of UV light. Live cells were selected based upon both forward vs. side-scatter profiles and absence of PI or DAPI stain and ALDEFLUOR/DEAB treated cells were used to define negative gates for ALDH.