Wild type or irradiation resistant Daoy or HD-MB03 cells (106 cells) were softly dissociated with 1X accutase (HyClone HyQTase, Fisher Scientific). The cells were then labeled with a mouse monoclonal PE-conjugated anti-CD146, (BioCytex, Marseille, FRANCE). Fluorescence was measured using the 488 and 633 nm lasers of a flow cytometer (FACS Canto - BD Biosciences, FRANCE). The gating strategy of these experiments is described in Supplementary Fig. S7 . The data were collected with the BD FACSDiva software application. Percentages of CD146-labeled isolated cells were calculated with FlowJo® software version 18.0 (Flexera, Hamburg, GERMANY).
Pe conjugated anti cd146
Manufactured by Biocytex
PE-conjugated anti-CD146 is a monoclonal antibody that binds to the CD146 cell surface antigen. CD146 is a transmembrane glycoprotein involved in cell-cell adhesion. The PE (Phycoerythrin) fluorescent dye is conjugated to the antibody, enabling detection and quantification of CD146-expressing cells using flow cytometry.
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2 protocols using pe conjugated anti cd146
1
Flow cytometry analysis of CD146+ cells
2
Multiparametric Flow Cytometry Immunophenotyping
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The cells were harvested as mentioned above and resuspended in PBS containing 1% FBS along with 0.5 mM EDTA. The cell suspension was incubated with specific antibodies for 1 h at 4°C. The following monoclonal antibodies were used: FITC (fluorescein isothiocyanate)-conjugated anti-CD 13 (ID Labs, # IDAC1071), FITC-conjugated anti-CD31 (Immunostep, # 31F-100T), FITC-conjugated anti-CD44 (BD Biosciences, # 560977), FITC-conjugated anti-CD 49b (BD Biosciences, # 555498), PE (phycoerythrin)-conjugated anti-CD 45 (BD Biosciences, # 555483) and PE-conjugated anti-CD146 (Biocytex, # 5050-PE100T). Thereafter, the cells were washed with PBS and fixed in 2% PFA. Analysis was performed on at least 10,000 events per sample using a FACS caliber flow cytometer (BD Biosciences). The acquisition was performed using CELLQUEST software (BD Biosciences) and analyzed using FCS-express software (DeNovo).
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