EFB Dengue Literature Review

FACS Sorting

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FACS Sorting

Overview

Fluorescence-activated cell sorting (FACS sorting) uses the same principles as analytical flow cytometry but physically separates cells into collection tubes based on defined surface phenotype gates. In B cell immunology, FACS sorting is used to isolate specific subsets (e.g., plasmablasts, DN memory B cells, switched memory B cells) for downstream functional, transcriptomic, or molecular analysis — most commonly BCR sequencing, ELISpot, or single-cell RNA-seq.

Key Points from Literature

  • Wei et al. used a BD FACSAria to sort CD19⁺CD20⁺IgG⁺IgD⁻ cells into CD27⁺ and CD27⁻ (DN) fractions from peripheral blood of a healthy donor and an SLE patient for VH3 family BCR sequencing — establishing the somatic hypermutation profiles of both populations (see Wei2007 - DN Memory B Cells in SLE).

  • Tonsillar B cells were pre-enriched by SRBC rosetting before staining and sorting in the same study (see Wei2007 - DN Memory B Cells in SLE).

  • Magnetic bead-based negative selection (Miltenyi B Cell Isolation Kit II) was used to enrich total B cells from PBL prior to CpG proliferation assays; tonsil B cells were further depleted of CD10⁺ and CD27⁺ cells by MACS before fractionating DN from naive cells on IgD expression (see Wei2007 - DN Memory B Cells in SLE).

  • Tipton2015 multi-population sort (FACSAria II): Four to five populations simultaneously sorted from the same PBMC preparation: (1) IgD⁺CD27⁻ resting naive, (2) acN cells (IgD⁺CD27⁻MTG⁺CD24⁻), (3) IgD⁻CD27⁺ memory, (4) CD138⁻ ASCs, (5) CD138⁺ ASCs. ~10⁴–3×10⁵ cells collected per population; all populations taken to NGS for simultaneous connectivity analysis. Also included single-cell sorting of 9G4⁺ plasmablasts into 96-well plates for monoclonal antibody generation (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Jenks2018 DN1/DN2 sort (FACSAria II): Multiple B cell subsets sorted simultaneously: rNAV (IgD⁺CD27⁻ resting naive), aNAV (IgD⁺CD27⁻CXCR5⁻CD19^hiCD21⁻), SWM (IgD⁻CD27⁺), DN1 (IgD⁻CD27⁻CXCR5⁺), DN2 (IgD⁻CD27⁻CXCR5⁻CD11c⁺CD19^hi), and total NAV from SLE patients and healthy controls. Sorted populations used for: RNA-seq (10,000–50,000 cells), ATAC-seq (10,000–50,000 cells), BCR sequencing, and in vitro differentiation cultures. The CXCR5 gate is the critical discriminator for DN1 vs. DN2 sort purity (see Jenks2018 - DN2 B Cells and EF Pathway in SLE).

  • Scharer2019 multi-subset sort for multi-omic profiling (FACSAria II): Five B cell subsets + ASCs sorted simultaneously from SLE patients and healthy controls: resting naive (rN: IgD⁺CD27⁻CXCR5⁺CD21⁺), T3 transitional (MTG⁺CD24⁺ within IgD⁺CD27⁻), activated naive (aN: IgD⁺CD27⁻CXCR5⁻CD19^hiCD21⁻), isotype-switched memory (SM: IgD⁻CD27⁺), DN2 (IgD⁻CD27⁻CXCR5⁻CD11c⁺CD19^hi), and ASCs (IgD⁻CD27^hiCD38^hi). Each sorted population was split for three downstream assays: RRBS (DNA methylation), ATAC-seq (chromatin accessibility), and RNA-seq (transcriptomics). This is the most comprehensive multi-omic sort design applied to human B cell subsets (see Scharer2019 - Epigenetic Programming in SLE B Cells, n=9 SLE + 12 HC).

  • ASC and naive B cell sorting for V(D)J sequencing: Total ASCs gated as CD3⁻CD14⁻CD16⁻CD19⁺CD38⁺CD27⁺ single live cells; naive B cells gated as CD3⁻CD14⁻CD16⁻CD19⁺CD27⁻IgD⁺CD38⁺ single live cells. Sorted on a three-laser BD FACS. For bulk sequencing, B cells were pre-enriched by negative selection (StemCell Pan-B) followed by CD138⁺ positive selection (Miltenyi beads) to enrich mature ASCs (see Woodruff2020 - EF B Cell Responses in COVID-19).

  • Single-cell and pool sorting of dengue PBs and antigen-specific MBCs (FACSAria): Appanna2016 sorted two populations at different timepoints from the same patients: (1) Plasmablasts (CD19⁺CD20⁻CD27^hiCD38^hi) from acute phase (days 3–7) — single cells into 96-well plates for Sanger sequencing and mAb cloning; (2) DENV-specific MBCs (CD19⁺CD20⁺CD27⁺, gated by binding to Alexa Fluor-labelled live DENV-1, -2, -3 particles) from convalescence (days 16–166) — both single-cell and pool sorts. Pools were taken to 454 sequencing. This is the first dengue study combining antigen-specific B cell sorting with BCR sequencing for longitudinal clonal tracking (see Appanna2016 - Plasmablasts as Subset of Memory B Cell Pool, FACSAria, n=12 dengue).

Contradictions & Debates

None documented in current wiki sources.

Conventional Flow Cytometry, BCR Sequencing, RNA Sequencing, ATAC-seq, ELISpot, Activated Naive B Cell, DN2 B Cell, Double-Negative B Cell

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