Cd44 pe

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.

Colony-forming cells from SF were detached at passage 2 with TrypLE (Thermo Fisher Scientific) and suspended in FACS buffer (2% FBS and 5 mM EDTA in PBS). The cells were stained with the CD34-PerCP (Novus), CD44-PE (R&D Systems, Minneapolis, MN, USA), CD45-FITC (BD Biosciences, San Jose, CA, USA), CD90-PE-Cy7 (BD), CD105-APC (Novus), and Ghost Dye Violet 510 for dead cells (Tonbo Biosciences, CA, USA). For CD73, a mouse anti-CD73 antibody (BD) was used, followed by the secondary antibody conjugated with Alexa Fluor 488 (Abcam).
The synovial tissue was analyzed by mincing the synovium and digesting it for 3.5 h at 37 °C with 0.4 mg/mL Liberase (Roche Diagnostics, Mannheim, Germany) and 0.1 mg/mL DNase (Sigma-Aldrich, St Louis, MO, USA). Residual red blood cells were lysed with ACK Lysing Buffer (Thermo Fisher Scientific), the cells were passed through a 70 μm strainer (Greiner Bio-One GmbH, Frickenhausen, Germany) to yield single-cell suspensions. The cells were then stained with the CD44-PE, CD73-PE-Cy7 (Bioss), and CD90-APC (BD), as well as with Ghost Dye Violet 450 (Tonbo Biosciences) to identify dead cells. The proportion of antigen-positive cells was evaluated using a FACSVerse instrument (BD).