Alexa fluor 488 conjugated goat anti mouse igg1 secondary antibody

BLS-DR2a or BLS-DR2b cells were plated onto poly-L-Lysine (Sigma-Aldrich) -coated slides. The fixation, permeabilization, and blocking of nonspecific binding sites of the cells were performed using the Image-iT® Fix-Perm Kit (Invitrogen, California, USA) according to the manufacturer’s instructions. The cells were then incubated with 10 μg/ml primary anti-DR2a or anti-DR2b antibody overnight at 4°C. Following several wash steps with PBS + 0.5% BSA, the cells were incubated with 1 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG1 secondary antibody (Invitrogen) to target anti-DR2a antibody or 10 μg/ml Alexa Fluor 647-conjugated goat anti-mouse IgG2b secondary antibody (Invitrogen) to target anti-DR2b antibody for 1 h at room temperature. Thereafter, cells were washed several times with PBS + 0.5% BSA and mounted with SlowFade Diamond Antifade Mountant with DAPI (Invitrogen) and coverslips. The images were captured using fluorescence microscopy (Zeiss, Oberkochen, Germany) and analyzed using ImageJ (NIH).

The binding affinity of the antibody to the cell surface expressing c-Met was determined by flow cytometry analysis. Approximately 1 × 10⁶ cells were harvested from a monolayer culture, washed, and incubated with a saturating amount (10 μg/mL) of primary antibody in PBS (pH 7.4) with 2% FBS (staining buffer) for 30 min on ice. The cells were then washed and stained with an Alexa Fluor 488–conjugated goat anti-mouse IgG1 secondary antibody (diluted 1:100; Invitrogen, USA) for 15 min on ice. The cells were subsequently incubated with a fluorescein-conjugated mAb for 30 min on ice. The cell suspension was washed three times with PBS and then analyzed using a FACS Calibur flow cytometer (BD Immunocytometry Systems, NJ, USA).