Mouse anti human cd44 antibody

Western blotting was performed as previously described [22 (link)]. Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China). Secondary antibodies included an HRP-conjugated goat anti-rabbit antibody. GADPH was used as a loading control.

The SCC-9 cells (CRL-1629) (American Type Culture Collection [ATCC], VA, USA) were grown in Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS). When the cell density reached 80-90%, the cells were digested by trypsin (Gibco, Grand Island, NY, USA) for single cell suspension. The cells were then centrifuged at 1500 rpm for 10 mins, and washed twice with PBS, and resuspended in 200μL PBS. Next, the cells were incubated with 100μl mouse anti-human CD44⁺ antibody (Cell Signaling Technologies (CST), Beverly, MA, USA) for 45 min. The cells were washed twice by PBS and resuspended in 200μL PBS. The SCC-9 cells were separated by flow cytometry (FACSCanto II) (BD Biosciences Company, NJ, USA) based on CD44 immunofluorescent labeling. The CD44⁺ cells corresponded to OSCC cells whereas the rest were non-stem-like cells. These cells were preserved and frozen for further use or used for further experiments.