Anti cxcr4

GBM cells were resuspended in FACS flow buffer (BD Biosciences, San Jose, CA, USA) with DAPI (for exclusion of dead cells; 1:1000) before flow cytometry analysis using FACS Canto II (BD Biosciences). In order to identify autofluorescent (Fluo⁺) cells, GBM cells were excited with a 488 nm blue laser and selected as the intersection with filters 530/40 and 580/30 (Figure S1A).
To characterize autofluorescent cells, human primary GBM cultures (GBML1, GBML18, and GBML42) were analyzed by flow cytometry for the expression of CSCs surface markers. Briefly, 1 × 10⁵ cells were incubated with the suitable dilution of appropriate isotype-matched control or specific antibody in 100 µL of PBS for 30 min at 4 °C in the dark. Antibodies used were anti–CD133/1 (1:10; Miltenyi Biotec, Madrid, Spain), anti–CD15 (1:10; BD Biosciences), and anti–CXCR4 (1:10; BD Biosciences). All antibodies were APC–conjugated. Cells were resuspended in 200 µL of FACS flow buffer (BD Biosciences) with DAPI and analyzed by FACS Canto II. Data was analyzed with FlowJo 10.0 software.

Immunophenotyping of 82.3 cells was performed by flow cytometric analysis. Cells were washed in 1× PBS containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MO, USA) and 0.01% sodium azide. For intracellular markers, cells were permeabilized with fix and perm reagent (BD Italia) following the manufacturer’s instructions. The following antibodies were used: FITC (Fluorescein isothiocyanate) conjugated mouse mAbs anti-CK7 and anti-CK19 (Abcam, Cambridge, UK) and anti-CD44 (BD Bioscience, San Jose, CA, USA), APC-(allophycocyanin) conjugated mouse mAbs anti-EPCAM and anti-AFP (BD Bioscience Europe), anti-CD34 and anti-CD133 (Miltenyi Biotec S.r.l., Bologna, Italy), PE (phycoerytrin) conjugated mAbs anti-CD24, anti-CXCR4, anti-Oct3/4, anti-FOXA1/2, anti-PDX1, (all from BD), anti-CD338 (R&D Systems, Inc., Minneapolis, MN, USA), PerCP-Cy 5.5 conjugated mAbs anti-SOX2/17, Alexa Fluor 647 conjugated mAbs anti-Nanog, anti-Stro1, and Alexa Fluor 488 conjugated mAbs anti-PAX6 (BD Bioscience Europe).

For flow cytometry analysis organoids were dissociated in a 10:1 mixture of Accutase (Sigma-Aldrich, A6954) and 10× Trypsin (Gibco, 15400) shaking at 37 °C and filtered through a 35 µm strainer. Aliquots were counted on an Invitrogen Countess II cell counter. Antibody staining was performed in DMEM/F12 + 4% BSA using the following antibodies: anti-NCAM (BD Biosciences, 564058), anti-CXCR4 (BD Biosciences, 560936) and anti-TRA-1-60 (BD Biosciences, 563188).

Stem cell markers in rhabdomyosarcoma cells were evaluated by staining with monoclonal antibodies conjugated with phycoerythrin (PE) anti–CD133 anti–CD90, anti–CXCR4, anti−CD105, and with allophycocyanin (APC) anti-CD117(all from BD Biosciences, Buccinasco, Italy).
Appropriate isotype controls for non-specific binding were used for each antibody. A minimum of 50,000 events were acquired for each sample by a flow cytometer (FACSCalibur, BD Biosciences) using CellQuest software (BD Biosciences) for data acquisition and analysis.