Anti igm pe cy7 il a30

PBMCs isolated from the two cattle that were inoculated with the anti-bovine PD-L1 antibodies were labeled with CFSE and cultured for 6 days with heat-inactivated FLK-BLV supernatants in triplicate. Medium only and heat-inactivated supernatants of FLK cells were used as a negative control. Following cultivation, the PBMCs were stained using anti-CD4-Alexa Fluor 647 (CC30; Bio-Rad), anti-CD8-PerCp/Cy5.5 (CC63; Bio-Rad) and anti-IgM-PE/Cy7 (IL-A30; Bio-Rad). CC30 was pre-labeled with Alexa Fluor 647 using Zenon Mouse IgG1 Labeling Kits (Thermo Fisher Scientific), and CC63 and IL-A30 was pre-labeled using the Lightning-Link PerCp/Cy5.5 Conjugation Kit (Innova Biosciences) and the Lightning-Link PE/Cy7 Conjugation Kit (Innova Biosciences), respectively. The cells were immediately analyzed by flow cytometry, as described above.

The binding of 4G12 to bovine PD-L1 on circulating IgM⁺ B cells was investigated to calculate the PD-L1 occupancy of 4G12 following inoculation. PBMCs isolated from #368 were pre-incubated with 10 μg/ml rat IgG2a isotype control (BD Bioscience) or 4G12, and then incubated with APC-conjugated anti-rat Ig (Beckman Coulter) at room temperature for 20 min. The same secondary antibody pre-incubated with IgG from rat serum (Sigma-Aldrich) at 37°C for 15 min was used as an unstained control. After washing twice, anti-IgM-PE/Cy7 (IL-A30; Bio-Rad) was reacted at room temperature for 20 min. The binding of the antibodies was then detected by flow cytometry, as described above. PD-L1 occupancy was estimated as the percentage of the in vivo binding (indicated as the number of cells positively stained by rat IgG2a isotype control) occurred at the total available binding sites (indicated as the number of cells positively stained by a saturated concentration of 4G12).