Goat anti rat ig fitc

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).

Cells were fixed in normoxic (21% O₂) or hypoxic (1% or 5% O₂) conditions for 5 minutes in 500 μL of flow cytometry fixation buffer (R&D Systems) and washed in stain buffer containing BSA (BD Pharmingen). Cells were resuspended in 100 μL of stain buffer and incubated for 45 minutes without antibodies or with nonspecific fluorescence labeled antibody (Mouse IgG1 κ Isotype Control-Alexa Fluor 647, BD Pharmingen) at 1:20 dilution, with fluorescence labeled antibody against integrin α5 (Mouse Anti-Human CD49e-Alexa Fluor 647, BD Pharmingen) at 1:20 dilution, with primary nonspecific antibody (Purified Rat IgG2a κ Isotype Control, BD Pharmingen) at 1:25 dilution or with primary antibody against integrin β1 (Purified Rat Anti-Human CD29, BD Pharmingen) at 1:25 dilution followed by incubation for 45 minutes with fluorescence labeled secondary antibody (Goat Anti-Rat Ig-FITC, BD Pharmingen) at 1:50 dilution. Cells were washed with stain buffer and resuspended in 1 mL of stain buffer. Flow cytometry was performed using the BD LSRFortessa^TM (BD Biosciences).