Livedead ef780

biotinylated SARS-CoV-2 S protein and RBD were conjugated to streptavidin-bound fluorophores. Briefly, the biotinylated proteins were incubated for a minimum of 1 h at 4°C with the streptavidin-conjugates AF647 (Biolegend), BV421 (Biolegend) and BB515 (BD Biosciences) at a 1:2 protein to fluorochrome ratio. The fluorescent probes were incubated for at least 10 min with 10mM biotin (GeneCopoeia) to saturate the unconjugated streptavidin-fluorochrome complexes. Cryopreserved macaque PBMCs from 2 weeks after final immunization (week 12) were thawed and counted. 5x10⁶ cells were stained for 30 min at 4°C with the fluorescent probes, a viability marker (LiveDead-eF780, eBiosciences) and the following B cell-specific antibodies: anti-CD20-PE-CF594 (clone 2H7; BD Biosciences), anti-IgG-PE-Cy7 (clone G18-145;BD Biosciences), anti-CD27-PE (clone M-T271; BD Biosciences), and anti-IgM-BV605 (clone MHM-88; Biolegend). Cells were washed twice with FACS buffer and acquired on the FACS-ARIA-SORP 4 laser (BD Biosciences). Analysis was performed on FlowJo v.10.7.1.

To generate probes for B cell sorting, biotinylated GPC-I53-50A from lineage II and IV as well as a biotinylated HCV E1E2-I53-50A (K.S. et al., unpublished data) were conjugated to the streptavidin-bound fluorophores AF647, BV421, and BB515, respectively. Conjugation was performed by incubating the biotinylated proteins for a minimum of 1 h at 4°C with the streptavidin-conjugates at a 1:2 protein to fluorochrome ratio. To saturate unconjugated streptavidin-fluorochrome complexes the fluorescent probes were next incubated for at least 10 min with 10mM biotin (GeneCopoeia) to saturate the unconjugated streptavidin-fluorochrome complexes. Week 29 rabbit PBMCs were then counted and 5x10⁶ cells were stained for 30 min at 4 with the fluorescent probes, a viability marker (LiveDead-eF780, eBiosciences), and a rabbit PE-conjugated anti-IgG marker (Biolegend). Prior to their acquisition on the FACS-ARIA-SORP 4 laser (BD-Biosciences), cells were washed twice with FACS buffer. Viable IgG+ B cells that were negative for HCV E1E2-I53-50A and showed dual staining for both GPC-I53-50A from lineage II and IV were sorted. Analysis was performed on FlowJo v.10.7.1.

KOLF2 iPSCs, genetic variant and normal, were stimulated to differentiate into neural progenitor cells with STEMdiff SMADi Neural Induction Kit (STEMCELL, Vic, Australia). At day 0, and 24 of neural cell differentiation 5 × 10⁵ cells were collected, LIVEDEAD ef780 stained for 10 min (eBioscience, US), fix/permeabilized according to Transcription factor staining buffer set (eBioscience, US) and stained for stem cell expression marker (OCT3/4), and neuronal cell marker (PAX6). Samples were collected using an LSRII X20 flow cytometer (BD, Biosciences), and analysed with FlowJo software (TreeStar Inc, Ashlan, OR, USA). Gating strategy was total cells, with subsequent gating on live cells and then singles, prior to determination of percentage frequency protein marker expression. Statistical analysis of data was with one-way ANOVA and Bonferroni’s correction for multiple testing performed using GraphPad Prism Version 8 software (GraphPad Software Inc., La Jolla, CA, USA). In addition, cells were collected for RNA extraction (RNeasy Minikit, with DNase on column treatment; Qiagen).