Mouse anti human cd44 antibody

The following reagents and kits were used: cell fixation buffer and Alexa Fluor 488 anti-human CD24 antibody (BioLegend), cell permeabilization kit (MiltenyiBiotec), mouse anti-human CD45 conjugated with Alexa Fluor 488 and Hoechst33342 (Invitrogen), Pho-4EBP1 Thr37/46 antibody, vimentin antibody conjugated with Alexa Fluor 647 and pho-GSK3β Ser9 antibody (Cell Signaling Technology), mouse anti-human CD44 antibody (BD Biosciences), Pho-S6K1 Ser424 antibody, pan cytokeratin C11 antibody, the donkey anti-rabbit and anti-mouse IgG H&L conjugated with Alexa Fluor 488 and 647 secondary antibodies (Abcam), pan cytokeratin AE1/AE3 antibody (Novus Biologicals), LKB1 antibody, goat anti-rabbit and anti-mouse IgG(H + L) superclonal secondary antibodies, HRP conjugated (Thermo Fisher), everolimus (Selleckchem), 10X TGS buffer and 10X TG buffer for western blot (Omics Bio), and MTT powder (Sigma-Aldrich).

Cell culture and gene silencing procedures were performed as mentioned above. Cells were stained using a standard flow cytometry protocol for the detection of surface CD44 antigen using V450 Mouse Anti-human CD44 antibody according to the manufacturer’s instructions (BD Biosciences, Franklin Lakes, NJ, USA, Clone# G44-26, Cat# 561292). Flow cytometry analysis was performed using a MACSQuant analyzer 10 (Miltenyi Biotec, Auburn, CA, USA) and FlowJo analysis software (FlowJo, Ashland, OR, USA).

CD44-APC Mouse Anti-Human CD44 antibody (Clone G44-26, BD Pharmingen™, Heidelberg, Germany) and APC Mouse IgG2b κ Isotype Control (Clone 27-35, BD Pharmingen™) were added to a final dilution of 1:10 to PBS-washed cells. The concentration of cells was adjusted to a range from 1 × 10⁶ to 1 × 10⁷ cells per 100 µL PBS before adding the antibodies. The suspension was incubated for 45 min at 4 °C in the dark. After incubation, the cells were washed twice with PBS by repeating centrifugation at 300× g for 8 min and adding PBS. The cells were suspended in 500 µL PBS for flow cytometry cell sorting (FACS) analysis.
Cells tagged with the CD44-APC antibody were FACS sorted. The cell suspensions were filtered first to obtain single cells for FACS. Each FACS analysis was performed by gating for single cells, then for living cells via DAPI labeling. Then, the cells were sterile filtered through a 20 μm sieve for the collection of unsorted, CD44-low and CD44-high populations directly into PBS for the subsequent experiments.