Mouse anti bovine cd21 rpe

Antibody staining of cells and flow cytometric quantification of T and B cells were described in detail by Liermann et al. (28 (link)). Briefly, cells were stained by monoclonal antibodies for cluster of differentiation (CD)2 (T cells) (mouse anti-bovine CD2: FITC; Bio-Rad Laboratories, Hercules, USA), CD4 (T helper cells) (mouse anti-bovine CD4: FITC; Bio-Rad), CD8 (cytotoxic cells) (mouse anti-bovine CD8: Alexa Fluor^® 647; Bio-Rad), and CD21 (B cells) (mouse anti-bovine CD21: RPE, Bio-Rad) or the corresponding isotype controls (mouse IgG1 negative control: FITC; mouse IgG2a negative control: FITC; mouse IgG2a negative control: Alexa Fluor^® 647; mouse IgG2b negative control: RPE; Bio-Rad). A flow cytometer Type GalliosTM (Beckman Coulter GmbH, Krefeld Germany) was used.

PBMCs were isolated from the heparinized blood samples of the bovine with HISTOPAQUE 1.083 (Sigma-Aldrich, USA) according to the manufacturer’s instructions. The PBMCs were then used to identify FMDV serotype O and serotype A cross-reactive plasmablasts. Purified FMDV (O/Tibet/99) inactivated 146S antigen was labeled with Lightning-Link Rapid FluoProbes 647H (Innova Biosciences, San Diego, USA) according to the manufacturer’s instructions. Purified FMDV (A/WH/CHA/09) inactivated 146S antigen was labeled with Pacific Blue (Thermo Scientific, USA) according to the manufacturer’s instructions. Freshly isolated PBMCs were stained with O-FMDV 146S-FluoProbes 647H, A-FMDV 146S-Pacific Blue, mouse anti-bovine CD21-RPE (Bio-Rad, USA) and mouse anti-bovine IgM-FITC (Bio-Rad, USA) for 30 min at 4°C in PBS containing 2 mM EDTA and 0.5% BSA. The parallel staining of PBMCs that lacked O-FMDV 146S-FluoProbes 647H and A-FMDV 146S-Pacific Blue was used as fluorescence minus one (FMO) control. The stained samples were immediately analyzed by flow cytometry, and one million PBMCs were acquired for counting the proportion of FMDV serotype O and A cross-reactive plasmablasts.