Anti human mouse cd44 fitc

Anti-Human/Mouse CD44 FITC (eBioscience) and Anti-Human CD24 PErCP-eFluor 710 (eBioscience), Anti-Human CD24 PE (eBioscience) and Anti-Human CD326 (EpCAM) PerCP-eFluor 710 (eBioscience) were used for flow cytometric analysis. Cells were harvested by trypsinization and washed once in cold PBS. Next, cells were counted and resuspended in cold PBS at a concentration of 1 × 10⁶ cells per 100 μl. Staining was performed by incubating 100 μl cells with 5 μl antibody on ice for 30 min.

After lentiviral transduction, cells were treated with or without TMZ and cultured for additional 48 h. After that, cells were collected and resuspended in binding buffer containing rh Annexin V-FITC for 10 min at RT. Then the cells were washed and binding buffer was added with propidium iodide (PI, 20 μg/ml), and FACS analysis was performed by BD FACSCalibur (BD Biosciences Inc., Franklin, NJ, USA) according to the Annexin V-FITC Apoptosis Detection Kit (eBioscience, Waltham, MA, USA). For CD44⁺ cell proportion, anti-Human/Mouse CD44 FITC (eBioscience) was used for FACS analysis. In addition, cell apoptosis was also quantified by Hoechst 33258 (5 μg/ml, Sigma-Aldrich, Darmstadt, Germany) and visualized through fluorescence microscope (Olympus, Tokyo, Japan). The images were analyzed with the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).