Cd44 apc

Tumors were resected from mice, dissociated by collagenase and hyaluronidase (StemCell Technologies, Vancouver, BC, Canada), incubated in ACK red blood cell lysis buffer (Invitrogen), and filtered through a sterile 40 µm cell strainer. ALDH enzymatic activity was stained using Aldefluor Kit (StemCell Technologies) or AldeRed ALDH Detection Assay (MilliporeSigma, Burlington, MA, USA). Briefly, 1 × 10⁶ cells were incubated with activated ALDH substrate or the equivalent volume of ALDH inhibitor diethyl aminobenzaldehyde (DEAB). DEAB controls were included for all treatment conditions. Cells were rinsed with PBS and stained for CD44 with either CD44-PE or CD44-APC (R&D Systems, Minneapolis, MN, USA) for 15 min at 4 °C. Human cells were identified by anti-HLA-ABC (PE; BD Pharmingen, NJ, USA). Viable cells were stained with DAPI (Molecular Probes, Eugene, OR, USA). For cell sorting, ALDH^highCD44^high CSC population was sorted against the remaining bulk tumor cells (i.e., ALDH^highCD44^low, ALDH^lowCD44^high, and ALDH^lowCD44^low). All flow cytometry analyses were conducted in a BD LSRFortessa flow cytometer (BD Biosciences). Results were analyzed with FlowJo software (LLC; Ashland, OR, USA) in triplicate wells per condition.

8505C cells were detached using trypsin, washed with PBS, and incubated for 30 minutes with CD44-APC (RD Systems). Living cells were identified using Hoechst stain (Invitrogen). Flow cytometry was performed on a FACS DiVa Machine (Becton-Dickinson, San Jose, CA, USA).