Immunophenotyping of Antigen-Specific B Cells and T Follicular Helper Cells

For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then surface stained with the relevant probes (APC or PE labelled stem, HEL, OVA or gp120 proteins for baiting antigen-specific GC B cells) [10 (link)] and the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend). For detection of Tfh cells ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend). For Ag-specific Tfh identification [23 (link)], cells were cultured as described above and then stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

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Jiang W., Pilkington E.H., Kelly H.G., Tan H.X., Juno J.A., Wheatley A.K, & Kent S.J. (2020). Aggregation by peptide conjugation rescues poor immunogenicity of the HA stem. PLoS ONE, 15(11), e0241649.

Publication 2020

Aqua Viability Dye CD45 APC-Cy7 CD3 BV785 F4/80 BV785 Streptavidin BV785 B220 BUV737 IgD BUV395 CD38 PE-Cy7 GL7 AF488 Live/dead Red B220 BV605 CD3 BV510 CD4 BUV737 CXCR5 BV421 PD-1 BV786 CD25 BB515 OX40 PeCy7 BD FACS Diva

Antibodies Antigen B cells Cd25 Cells Clone Cxcr5 Gp120 Mice Ox40 Proteins Stem Streptavidin

Corresponding Organization : Monash University

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Variable analysis

independent variables

Antigen used for baiting antigen-specific GC B cells (APC or PE labelled stem, HEL, OVA or gp120 proteins)
Cells cultured as described above for Ag-specific Tfh identification

dependent variables

Detection of antigen-specific GC B cells
Detection of Tfh cells ex vivo
Identification of Ag-specific Tfh

control variables

Aqua Viability Dye (Thermo) for live/dead staining
Fc blocking with anti-CD16/32 antibody (clone 93; BioLegend)
Surface staining with the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend)
For detection of Tfh cells ex vivo: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend)
For Ag-specific Tfh identification: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend)

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