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).

  • Antigen-specific B cell sorting with tetramers for BCR analysis: Sutton2021 used FACS sorting with Plasmodium falciparum circumsporozoite protein (PfCSP) and hemagglutinin (HA) tetramers to isolate antigen-specific B cells for Smart-seq2 scRNA-seq (163 cells from 11 donors). This enabled BCR analysis specifically on antigen-experienced cells within defined transcriptomic clusters — complementing the 10x Chromium unbiased approach on total B cells (see Sutton2021 - Alternative Lineage B Cells in Vaccination and Infection, FACSAria, n=11 donors).

  • CD80/PD-L2 sorting resolves memory B cells by developmental origin — with a naming trap. Memory B cells are commonly sorted into DP (CD80⁺PD-L2⁺), SP (CD80⁻PD-L2⁺), and DN (CD80⁻PD-L2⁻) fractions. Validated against GC-specific genetic fate mapping, DP is enriched for GC-derived memory and “DN” consists almost exclusively of GC-independent early memory cells — though the mapping is imperfect, with a considerable proportion of DP cells arising non-GC (see Glaros2025 - Multilayered Identity of B Cell Memory, review, no original data, mouse). ⚠ This “DN” means CD80⁻PD-L2⁻ and is unrelated to this wiki’s IgD⁻CD27⁻ DN sort gate — see Atypical B Cell.

  • ★ No surface sort gate cleanly separates GC-derived from GC-independent memory. Fate mapping shows the surrogate criteria the field used — cell-surface markers, class-switch status, and SHM load — are each “insufficient to definitively distinguish” the two populations, whose phenotypes overlap substantially and whose transcriptomes differ only subtly (see Glaros2025 - Multilayered Identity of B Cell Memory, review). Any sorted “GC-derived memory” fraction in the literature is an enrichment, not a purification.

  • ABCs sorted per Hao criteria for BCR sequencing (Russell Knode et al. 2017). Sorted murine ABC heavy and light chains were sequenced to assess repertoire diversity and somatic mutation, finding germline V_H/V_κ usage largely congruent with the follicular pool alongside a substantial mutated fraction (see Cancro2020 - Age-Associated B Cells, review — no original data; mouse).

  • Sorting to a higher purity standard for a knockout control experiment. Mouse Flt3L-derived bone marrow Plasmacytoid Dendritic Cells were sorted CD11b⁻CD45R^high^ on a FACSAria to >98% purity — against >85% for the magnetically enriched human pDCs in the same study. The stricter standard is applied where the cells are the genetic control (TLR7⁻/⁻ and TLR9⁻/⁻) validating an inhibitor’s specificity, i.e. where contaminating wild-type cells would directly undermine the claim (see Wang2006 - Flavivirus Activation of pDCs and TLR7 Signaling, in vitro).

  • [2026-08-27] Sorting four DN subsets for bulk RNA-seq — a practical input-scale reference. 30 million fresh PBMCs, stained within 2 h of isolation, Fc-blocked (human TruStain FcX, 10 min RT), two-step surface stain (CXCR5 at 37 °C first, remaining markers at 4 °C, 30 min each), SYTOX AADvanced added immediately before sorting for dead-cell exclusion. Sorted on a BD Aria II SORP into RLT Plus buffer with β-mercaptoethanol to preserve RNA, stored at −80 °C. Four populations (DN1–DN4) were recovered per donor from this input, sufficient for SMART-Seq2 libraries at ~10 M reads each (n=4 donors) (see Allard-Chamard2023 - DN3 B Cells Infiltrate Inflamed Tissues, methods). See RNA Sequencing.

  • [2026-08-27] Compensation and tracking controls for a sort of this size: VersaComp antibody-capture beads for compensation, Rainbow 8-peak calibration beads to hold signal consistent across sorting batches (see Allard-Chamard2023 - DN3 B Cells Infiltrate Inflamed Tissues).

  • Sorting is what makes the in vitro system interpretable — three distinct sorts in one study. (1) Day-6 IgD^neg^CD27^neg^ B_DN_ cells sort-purified out of Th1 and Th2 co-cultures for RNA-seq; (2) 10,000 cells per condition sorted as ATAC-seq input; (3) SLE patient subsets sorted into T-bet^hi^ DN2, T-bet^lo^ B_N_, DN1 memory and conventional switched memory for the head-to-head differentiation assay. Sorted on a FACSAria (see Zumaquero2019 - IFN-gamma Programs T-bet-hi B Cells for ASC Differentiation, human, n=20 HD + n=40 SLE + in vitro reconstruction). The third sort is what licenses the paper’s strongest claim — that DN2 cells give ≥50-fold more ASCs than naive cells — because bulk culture could not have separated those starting populations.

  • Sort buffer chosen by downstream assay, and magnetic pre-enrichment before the sorter. In Song2022 CD4 T cells were first enriched by magnetic negative selection (EasyStep) before surface staining and sorting on a FACSAria; cells for adoptive transfer were sorted into complete RPMI and washed into sterile PBS, while cells for RNA and Ig sequencing were sorted directly into RNAprotect Cell Reagent. For RNA-seq, three separate sorts were performed on different days, each pooling the spleens of four to six mice per timepoint — the replicate structure behind every transcriptomic claim in the paper (see Song2022 - Tfh Outside Germinal Centers Drive T-bet CD11c B Cells, mouse, LCMV-Armstrong + influenza PR8).

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, Single-Cell RNA Sequencing, Early Memory B Cell, Memory B Cell, Plasmacytoid Dendritic Cell, DN3 B Cell

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