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    • B cell Flow Cytometry

    B cell Flow Cytometry

    We used spectral flow cytometry to evaluate B cell phenotypes and frequencies in healthy young adults and older adults from the Sound Life cohort, at homeostasis (day 0) and during early (day 7) and late (day 90) flu vaccine response timepoints. The flu vaccine samples were collected in the 2020-2021 season. Flow cytometry was performed using a 23 color panel of antibodies and dyes on a Cytek Aurora spectral flow cytometer. Antibodies, fluorophores, dyes, and source information are provided below:

    TargetFlurophoreCloneVendorCat.#
    CD10BV750HI10aBD Biosciences747517
    CD11cAF700Bu15BioLegend337220/337219
    CD183 (CXCR3)BV785G025H7BioLegend353737/353738
    CD19BV650HIB19BioLegend302238/302237
    CD20APC-Cy72H7BioLegend302314/302313
    CD21BUV6611048BD Biosciences750187
    CD23 (FcεRII)APCEBVCS-5BioLegend338514/338513
    CD24BV605ML5BioLegend311124/311123
    CD27BV421L128BD Biosciences742731
    CD32 (FcγRII)FITCFUN-2BioLegend303204
    CD38PE-Dazzle594HIT2BioLegend303538/303537
    CD45RB**PEMEM-55**BioLegend310204
    CD69BUV395FN50BD Biosciences564364
    CD3PE-Cy7*variousBD Biosciences563423
    CD7PE-Cy7*variousBioLegend343114/343113
    CD14PE-Cy7*variousBD Biosciences557742
    CD16PE-Cy7*variousBioLegend302015/302016
    CD33PE-Cy7*variousBioLegend366618/366617
    CD61PE-Cy7*variousBioLegend 336416/336415
    CD235aPE-Cy7*variousBioLegend 306619/306620
    CD73BUV805AD2BD Biosciences748584
    CD95 (FAS)BV711DX2BD Biosciences561633
    HLA-DRBUV737G46-6BD Biosciences748339
    IgA***PerCp-Vio700IS11-8E10Miltenyi130-113-478
    IgDPE-Cy5IA6-2BioLegend348249/348250
    IgGBUV496G18-145BD Biosciences741172
    IgMBUV563UCH-B1BD Biosciences74892
    ViabilityFVS510nanBD Biosciences564406

    * All PE-Cy7 antibodies are included in the "Lin/Dump" channel.
    ** MEM-55 antibody clone binds to a glyco-epitope of CD45RB
    *** We recommend excluding IgA from downstream analysis due to non-specific staining of cells 

    To analyze changes in cell type abundance across age cohorts, we gated specific B cell subpopulations. Cells were gated using a standard lymphocyte, singlet and live cell gating before reaching the first B cell gate (B_cells_filter1). The gating diagrams below show how B cell populations were identified:

    Gate NameX FeatureY FeatureQuantified Cell Type
    Live_cellsCD19Lin
    └ B_cells_filter1CD19CD20
      ├ plasma_cells_filter1CD38CD27
      ┆└ plasma_cells_filter2Plasma cell
      └ B_cells_filter2CD19CD11c
           ├ efector_B_cellsCD27CD11c
           ┆ ├ CD27_neg_effector_B_cellsCD27- effector B cell
           ┆ └ CD27_pos_effector_B_cellsCD27+ effector B cell
           └ not_effectorCD32CD23
              ├ t2_memory_B_cellsType 2 polarized memory B cell
              └ not_t2_memory_B_cellsCD27CD45RB
                ├ antigen_inexperienced_B_cellsCD24CD38
                ┆  ├ naive_B_cellsNaive B cell
                ┆  └ transitional_B_cellsTransitional B cell
                └ antigen_experienced_B_cellsCD27CD95
                    ├ CD95_memory_B_cellsCD95 memory B cell
                    └ not_CD95_memory_B_cellsCD27CD45RB
                        ├ Early_memory_BEarly memory B cell
                        ├ Core_memory_BCore memory B cell
                        └ Late_memory_BLate memory B cell

    Data from these flow cytometry experiments are provided below:

    1. A .tar archive file containing .fcs files from each sample suitable for analysis using Flow Cytometry software, along with a .csv table with metadata for each file. The .fcs files contain all events in the experiment, including those labeled as non-B cells.
    2. A compressed table (.csv.gz) file containing annotated b cell event data with scaled expression for each measured marker.
    3. A table (.csv) with the frequency of each B cell type in each sample, as the fraction of live events.

    The columns provided in the metadata table an CSV files are provided below:

    Sample Identifiers
    flow.fileName: The file name of the .fcs file
    cohort.cohortGuid: A Globally Unique Identifier (GUID) of the Cohort the subject enrolled in for our study
    subject.subjectGuid: A GUID for the Subject
    sample.sampleKitGuid: A GUID for the Sample Kit, representing all material collected at a visit

    Subject Metadata
    subject.biologicalSex: The biological sex of the Subject
    subject.birthYear: The Birth Year of the Subject
    subject.ageAtFirstDraw: The Age of the Subject at their first on-study sample collection
    subject.ageGroup: The Age Group of the Subject (Young Adult or Older Adult)
    subject.race: The self-reported Race of the Subject
    subject.ethnicity: The self-reported Ethnicity of the subject
    subject.cmv: The CMV Status of the subject, as determined by an HCMV assay
    subject.bmi: The BMI of the Subject

    Sample Metadata
    sample.visitName: The name of the study visit (i.e. time point)
    sample.drawYear: The year of the study visit (e.g. 2021)
    sample.subjectAgeAtDraw: The age of the Subject in years at the time of sample collection

    Subject Metadata
    Subject.ageGroup: The Age Group of the Subject (Young Adult or Older Adult)
    subject.subjectGuid: A GUID for the Subject
    subject.biologicalSex: The biological sex of the Subject
    subject.cmv: The CMV Status of the subject, as determined by an HCMV assay
    subject.race: The self-reported Race of the Subject
    subject.ethnicity: The self-reported Ethnicity of the subject

    Sample Metadata
    sample.sampleKitGuid: A GUID for the Sample Kit, representing all material collected at a visit
    sample.visitName: The name of the study visit (i.e. time point)
    sample.subjectAgeAtDraw: The age of the Subject in years at the time of sample collection

    Flow Cytometry Results
    flow.cellType: The B cell type assigned to the cell
    flow.frac_live_cells: The fraction of live cells

    Sample Metadata
    cohort.cohortGuid: A Globally Unique Identifier (GUID) of the Cohort the subject enrolled in for our study
    subject.subjectGuid: A GUID for the Subject
    sample.sampleKitGuid: A GUID for the Sample Kit, representing all material collected at a visit
    subject.ageGroup: The Age Group of the Subject (Young Adult or Older Adult)
    sample.visitName: The name of the study visit (i.e. time point)

    Annotation columns
    flow.cellType: The B cell type assigned to the cell.
    flow.isotype: The isotype of the B cell based on Ig detection (if available)
    CXCR3_pos: A boolean value (True or False) for CXCR3 expression
    CD69_pos: A boolean value (True or False) for CD69 expression

    Feaature columns
    These columns provide scaled expression values for each feature, and are only present in the annotated cell .csv.gz file. See above for additional antibody and fluorophore information.
    CD23
    CD20
    CD11c
    CD69
    IgG
    IgM
    CD21
    HLA_DR
    CD73
    CD27
    CD24
    CD19
    CD95
    CD10
    CD183
    CD32
    Viability
    CD45RB
    IgD
    Lin
    CD38
    IgA

    Sound Life B cell Flow Analysis
    File Name Description Download Link
    sound-life_bcell_fcs.tar FCS files and metadata
    sound-life_bcell_labeled-events.csv.gz B cell events with labels
    sound-life_bcell_population_fractions.csv Fractions of live events