Apc anti human cd24 antibody

Parent cells and enriched cells were incubated with LGR5 (#ab75735; Abcam) and an isotype control (#ab171870; Abcam) and subsequently with secondary antibodies (Alexa Fluor 488) for 30 min at room temperature and washed three times in phosphate buffered saline (PBS) after each reaction to detect the proportion of LGR5 expression. Using the method described above, LGR5⁺ and LGR5⁻ glioma cells were sorted from parent cells by FACS.
To explore the proportion of reported stem cell markers in both LGR5⁺ and LGR5⁻ glioma cells, the cells were incubated with APC anti-human CD133 antibody (#130–090-826; Miltenyi), APC anti-human CD44 antibody (#17–0441-82; eBioscience), APC anti-human CD24 antibody (#311118; BioLegend), APC anti-human CD90 antibody (#328114; BioLegend) or APC anti-human EpCAM (#328208; BioLegend) antibody for 30 min at room temperature. APC anti-human IgG (#409306; BioLegend) was used as an isotype control for these markers. All stained cell suspensions were detected or sorted using an AccuriC6 cytometer (BD Biosciences) and analyzed by FlowJo 7.6.1.

Cells were resuspended in 10% FBS/PBS to reach a concentration of 10⁷ cells per milliliter. Twenty microliters of the cell suspension was stained with various antibodies diluted in 10% FBS/PBS for 1 h. Subsequently, cells were washed with 2% FBS/PBS and resuspended in 10% FBS/PBS for flow cytometry analysis (FACS). Antibodies used for our FACS analyses include APC anti‐human CD24 antibody (Cat # 311117; Biolegend, San Diego, CA, USA), PE anti‐human CD29 antibody (Cat # 303003; Biolegend), APC anti‐human CD133 (Cat # 17‐1338‐41; eBioscience, San Diego, CA, USA), and APC anti‐human CD49b (integrin alpha‐2; Cat # 17‐0500‐41; eBioscience).