Cd44 antibody

LAD2 MCs were incubated for 1 h in normoxia (21% O₂) or hypoxia (1% O₂) and fixed under the same conditions for 5 min in 500 μL of flow cytometry fixation buffer (R&D Systems). After fixation, MCs were washed in FACS buffer containing 1 × PBS, 1% BSA, and 0.1% sodium azide and were subsequently resuspended in 100 μL of this buffer. Next, MCs were incubated for 30 min with primary nonspecific antibody (mouse IgG1 isotype control, Novus Biologicals) at a 1:1000 dilution, with primary antibody against the RHAMM receptor (RHAMM/CD168 antibody, Novus Biologicals) at a 1:500 dilution followed by incubation in the dark for 30 min at 4 °C with fluorescence-labeled secondary antibody (goat anti-mouse IgG-FITC, Santa Cruz Biotechnology) at a 1:400 dilution, with primary nonspecific antibody (purified rat IgG2a κ isotype control, BD Pharmingen) at a 1:500 dilution, and with primary antibody against the CD44 receptor (CD44 antibody, Novus Biologicals) at a 1:500 dilution, followed by fluorescence-labeled secondary antibody (goat anti-rat Ig-FITC, BD Pharmingen) at a 1:500 dilution. Finally, MCs were washed with FACS buffer and resuspended in 1 mL of FACS buffer. Flow cytometry analysis was performed using a BD LSR Fortessa™ (BD Biosciences).

To determine the role of the CD44 receptor in MC adhesion to HA, we used an antibody that was suitable for inhibitory assays (CD44 antibody, Novus Biologicals) and an isotype control (purified rat IgG2a κ isotype control, BD Pharmingen). LAD2 cells were pretreated with 1 μg/mL or 10 μg/mL antibodies for 30 min before the adhesion assay. Concentrations were selected based on the manufacturer’s suggestions and the literature. After preincubation, an adhesion assay was performed as described above.