Identification of Influenza-Specific B Cells

HA-specific B cells were identified within a mean of 5.6 × 10⁶ cryopreserved PBMC (range 3.6–10.9 × 10⁶ PBMC/stain) by co-staining with HA probes conjugated to SA-PE or -APC (Thermofisher)34 (link)36 (link) and SA-BB515 probe control. Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), IgA-Vio450 (IS11-8E10) (Miltenyi), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend). Cell viability was assessed using Aqua Live/Dead amine-reactive dye (Thermofisher). An average of 1.38 × 10⁵ live CD19+ B cells (interquartile range 0.86–1.83 × 10⁵) were collected per subject per time point (155 samples in total) using a BD Fortessa configured to detect 18 fluorochromes and analysis was performed using FlowJo software version 9.5.2 (TreeStar).

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Wheatley A.K., Kristensen A.B., Lay W.N, & Kent S.J. (2016). HIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation. Scientific Reports, 6, 26478.

Publication 2016

CD19-ECD (J3-119) IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), BD Fortessa

Amine B cells Cell viability Fluorochromes Monoclonal antibodies Okt3 Stain

Corresponding Organization :

Other organizations : University of Melbourne, Peter Doherty Institute

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Variable analysis

independent variables

HA probes conjugated to SA-PE or -APC (Thermofisher)

dependent variables

Identification of HA-specific B cells

control variables

SA-BB515 probe control
Cell viability using Aqua Live/Dead amine-reactive dye (Thermofisher)

controls

Positive control: SA-BB515 probe control
Negative control: Not explicitly mentioned

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