Allophycocyanin conjugated anti mouse igm antibody

CHO/αIIbβ3 cells were transfected with various constructs using PolyJet^TM In Vitro DNA Transfection Reagent (SignaGen Laboratories) following the manufacturer’s recommendations. When needed, eptifibatide (Santa Cruz Biotechnology) was added to a final concentration of 20 μM after transfection. One day after transfection, the cells were detached with trypsin-EDTA, spin-washed twice with DMEM, and assessed for GFP expression via flow cytometry using either FACS Calibur or FACS Symphony A1 (BD Biosciences). Alternatively, to measure PAC1 binding, washed cells were incubated with 4 μg/mL of PAC1 (BD Biosciences) for 30 min at room temperature with agitation. Following two washes, the cells were subsequently incubated with allophycocyanin-conjugated anti-mouse IgM antibody (Thermo Fisher Scientific) for 30 min at 4°C, washed again, and analyzed by flow cytometry.