B cell activation experiments of the generated HCV-specific B cells were performed as previously described52 (link). In short, 4 million cells/mL in RPMI+ + were loaded with 1.5 μM of the calcium indicator Indo-1 (Invitrogen) for 30 min at 37 °C, washed with Hank’s Balance Salt Solution supplemented with 2 mM CaCl2 and 0.5% FCS, followed by another incubation of 30 min at 37 °C.
Antigen-induced Ca2+ influx of AR3C(gl) and HEPC74(gl) B cells was assessed on a LSR Fortessa (BD Biosciences) by measuring the 379/450 nm emission ratio of Indo-1 fluorescence upon UV excitation. Following 30 s of baseline measurement, aliquots of 1 million cells/mL were then stimulated for 250 s at RT with either H77 E2E1-I53-50A trimer, H77 E2E1-I53-50NP, UKNP4.1.1 E2E1-I53-50A trimer or UKNP4.1.1 I53-50NP. For the stimulation, trimers and nanoparticles were compared using equimolar amount of the E2E1-I53-50A component. Maximum Indo-1-fluorescence was determined by adding ionomycin (Invitrogen) to a final concentration of 1 mg/mL. Kinetics analyses were performed using FlowJo v8.1.
Antigen-induced Ca2+ influx of AR3C(gl) and HEPC74(gl) B cells was assessed on a LSR Fortessa (BD Biosciences) by measuring the 379/450 nm emission ratio of Indo-1 fluorescence upon UV excitation. Following 30 s of baseline measurement, aliquots of 1 million cells/mL were then stimulated for 250 s at RT with either H77 E2E1-I53-50A trimer, H77 E2E1-I53-50NP, UKNP4.1.1 E2E1-I53-50A trimer or UKNP4.1.1 I53-50NP. For the stimulation, trimers and nanoparticles were compared using equimolar amount of the E2E1-I53-50A component. Maximum Indo-1-fluorescence was determined by adding ionomycin (Invitrogen) to a final concentration of 1 mg/mL. Kinetics analyses were performed using FlowJo v8.1.
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