PBMCs (106) were stained for viability using the amine-reactive dye Aqua LIVE/DEAD (Invitrogen) followed by staining for surface markers. In addition to the tetramer probes rPfCSP-APC, (NANP)9-PE generated above, the staining panels included: anti-CD3-BV510 (BioLegend), CD8-BV510 (BioLegend), CD14-BV510 (BioLegend), CD56-BV510 (BioLegend), CD19-ECD (Beckman), CD27-QD605 (Invitrogen), CD21-Cy5PE (Becton Dickenson), CD38-Alexa Fluor 680 (Becton Dickenson), IgD-Cy7PE (Becton Dickenson), IgM-Cy5.5-PerCP (Becton Dickenson), and IgG- Cy7PE (Becton Dickenson). Cells were acquired and sorted using a BD FACS Aria II instrument (BD Immunocytometry Systems), and fluorescence-activated cell sorting (FACS) data was analyzed using FlowJo software (Tree Star). Gating strategy is shown in Fig. 1a . PfCSP-reactive (rPfCSP+ and/or (NANP)9+) CD19+CD27+IgG+ IgD−IgM− memory B cells were single cell, dry-sorted into 96-well PCR plates, rapidly frozen on dry ice and stored at −80°C until processing41 (link),42 (link).
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PfCSP-reactive Memory B Cell Isolation
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Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fred Hutch Cancer Center, Johns Hopkins University, Center for Infectious Disease Research, Massachusetts Institute of Technology, Broad Institute
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Variable analysis
independent variables
- Staining of PBMCs (10^6) with the amine-reactive dye Aqua LIVE/DEAD (Invitrogen)
- Staining of PBMCs for surface markers including: anti-CD3-BV510 (BioLegend), CD8-BV510 (BioLegend), CD14-BV510 (BioLegend), CD56-BV510 (BioLegend), CD19-ECD (Beckman), CD27-QD605 (Invitrogen), CD21-Cy5PE (Becton Dickenson), CD38-Alexa Fluor 680 (Becton Dickenson), IgD-Cy7PE (Becton Dickenson), IgM-Cy5.5-PerCP (Becton Dickenson), and IgG-Cy7PE (Becton Dickenson)
- Use of tetramer probes rPfCSP-APC and (NANP)9-PE
- Identification and isolation of PfCSP-reactive (rPfCSP+ and/or (NANP)9+) CD19+CD27+IgG+IgD-IgM- memory B cells
- Keeping PBMCs at 10^6 cells for staining
- Use of specified fluorophore-conjugated antibodies and tetramer probes
- Not explicitly mentioned
- Not explicitly mentioned
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