Sox2 fitc

Fluorescein-conjugated human antigen-specific or isotype control antibodies and anti-human IL-17RA PE were purchased from eBioscience; CD133 FITC and Nestin PerCPCy5.5 were from BD Bioscience, San Jose, CA; CD133 APC from Miltenyi Biotec; and Sox2 FITC was purchased from eBioscience. A published protocol was followed for surface and intracellular staining of the cells [79 (link)]. Tumor tissues obtained from 5 patients with histologically-verified glioblastoma were studied.

To analyze the apoptotic nature of the putative A549 stem cells, the Annexin V-PE Apoptosis Detection Kit I (559763; BD Biosciences, San Jose, CA, USA) was used. A total of 5 μL Annexin V-PE and 5 μL 7-AAD in 100 μL binding buffer were incubated with 1×10⁶ cells for 15 min at room temperature in the dark. Then the cells were washed twice with phosphate-buffered saline (PBS) and resuspended with 400 μL binding buffer. Flow cytometry was used for detection (Accuri C6, BD Biosciences). To analyze the relative CD133, ABCG2, OCT4, and SOX2 gene expression in the putative A549 stem cells, the cells were incubated with 100 μL PBS containing 5 μL CD133-PE (Miltenyi Biotec, Teterow, Germany) and 2.5 μL ABCG2-PE (Miltenyi Biotec) antibodies for 30 min in the dark. Then the cells were permeabilized in cold methanol and incubated with 2.5 μL OCT4-PE (eBioscience, San Diego, CA, USA) and 2.5 μL SOX2-FITC (eBioscience) antibodies for 30 min in the dark. The cells were washed twice with PBS and resuspended with 200 μL PBS for measurement by flow cytometry.