Alexa fluor 647 conjugated fab goat anti rat igg

To detect cell surface expression of the cytokine receptors mIL‑12Rβ2 and mIL‑23R, stably transduced Ba/F3-gp130 cells were washed with FACS buffer (PBS containing 1% BSA) and incubated at 5 × 10⁵ cells/100 μl FACS buffer supplemented with 2.5 μg of mIL‑23R mAbs (R&D Systems) or 1 μg of purified hamster mIL‑12Rβ2 mAbs (BD Biosciences) for 2 h on ice. After a single wash with FACS buffer, cells were incubated in 100 μl of FACS buffer containing a 1:100 dilution of Alexa Fluor 647–conjugated Fab goat anti-rat IgG (Dianova) or 0.25 μg PE mouse Armenian and Syrian hamster IgG cocktail (BD Biosciences) for 1 h at 4°C. Finally, cells were washed once with FACS buffer, suspended in 500 μl FACS buffer, and analyzed by flow cytometry (BD FACSCanto II flow cytometer; BD Biosciences). Data were evaluated using the FCS Express software (De Novo Software, Los Angeles, CA). Detection of surface expression of mIL‑12Rβ1 was achieved by incubating cells prepared as described with 100 μl of FACS buffer containing 10 μl of PE-conjugated IL‑12Rβ1 mAbs (R&D Systems) for 2 h at 4°C.