Indo 1

WEHI-231 B cell lines expressing the specific bNAbs (VRC01, PGT145, PGT121, and PG16) were donated by Takayuki Ota and David Nemazee at The Scripps Research Institute and were maintained in advanced Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FCS, β-mercapto-ethanol (55 µM), L-glutaMAX (2 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (advanced DMEM++). The activation experiments and calcium flux measurements were performed as described elsewhere^{26 (link),47 (link)}. In short, 1 day prior to the assay, cells were incubated with 1 µg/mL doxycycline to induce the expression of BCRs. The next day, cells were suspended at 4 million cells/mL in advanced DMEM++, labeled with 1.5 µM Indo-1 (Invitrogen) for 30 min at 37 °C and washed with HBSS (Hank’s Balance Salt Solution) containing 2 mM CaCl₂, followed by another incubation of 30 min at 37 °C. Aliquots of 1 million cells/mL were then stimulated at room temperature with SOSIP trimers, nanoparticles, or control reagents. Calcium (Ca²⁺) signals were recorded on a LSR Fortessa (BD Biosciences) by measuring for 210 s the 405/485 nm emission ratio of Indo-1 fluorescence upon UV excitation. ConM SOSIP.v7 and ConM SOSIP.v7-feritin were tested at concentrations of 50 nM Env equivalent, i.e., 6.25 nM of ferritin nanoparticles. Kinetic analyses were performed using FlowJo v10.

OT-1 cells (1 × 10⁷ cells) were prestained with anti-CD8β (clone eBioH35-17.2, cat 15266777, 1:400 dilution) in order to identify cell populations prior to loading with 2 µM Indo-1 AM (Life Technologies) for 40 min at 37 °C in PBS. Cells were resuspended in pre-warmed IMDM containing 1% FCS and supplemented with 2.5 mM CaCl₂ and 2.8 mM MgCl₂. The baseline Ca²⁺ levels were measured for 1 min before PE-labeled N4 dextramer (Immudex, Denmark) was added to stimulate the cells. Some samples were pretreated with TGFβ (5 ng/ml) 5 min before the addition of dextramer. Ionomycin (1 µg/ml) was added as a positive control for measuring saturated Ca²⁺ levels. Data are represented as the ratio of 398 nm (Indo-1 bound to Ca²⁺)/482 nm (unbound Ca²⁺) in OT-1 cells by flow cytometry and all data were acquired on an LSRII (BD Bioscience).

B cell activation experiments of the generated HCV-specific B cells were performed as previously described^{52 (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.

WEHI-231 B-cell lines expressing the specific bNAbs (VRC01, PGT145, and PGT121) were donated by Takayuki Ota and David Nemazee at The Scripps Research Institute and were maintained in advanced Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, β-mercapto-ethanol (55 µM), l-glutaMAX (2 mM), penicillin (100 U mL⁻¹), and streptomycin (100 µg mL⁻¹) (advanced DMEM++). B-cell activation experiments were performed as described elsewhere^{22 (link),65 (link)}. Briefly, 1 day prior to the assay, cells were incubated with 1 µg mL⁻¹ doxycycline to induce the expression of BCRs. The next day, cells were suspended at 4 million cells per mL in advanced DMEM++, labeled with 1.5 µM Indo-1 (Invitrogen) for 30 min at 37 °C and washed with Hank’s Balance Salt Solution containing 2 mM CaCl₂, followed by another incubation of 30 min at 37 °C. Aliquots of 1 million cells per mL were then stimulated at room temperature with SOSIP trimers, NPs, or control reagents. Doxycycline-induced WEHI-231 B cells were incubated with 5 μg mL⁻¹ of SOSIP trimers or the equimolar amount presented on SOSIP-ferittin or SOSIP-I53-50NPs. Calcium (Ca²⁺) signals were recorded on a LSR Fortessa (BD Biosciences) by measuring for 210 s the 405 per 485 nm emission ratio of Indo-1 fluorescence upon UV excitation. Kinetic analyses were performed using FlowJo v10.