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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:

    Target Flurophore Clone Vendor Cat.#
    CD10 BV750 HI10a BD Biosciences 747517
    CD11c AF700 Bu15 BioLegend 337220/337219
    CD183 (CXCR3) BV785 G025H7 BioLegend 353737/353738
    CD19 BV650 HIB19 BioLegend 302238/302237
    CD20 APC-Cy7 2H7 BioLegend 302314/302313
    CD21 BUV661 1048 BD Biosciences 750187
    CD23 (FcεRII) APC EBVCS-5 BioLegend 338514/338513
    CD24 BV605 ML5 BioLegend 311124/311123
    CD27 BV421 L128 BD Biosciences 742731
    CD32 (FcγRII) FITC FUN-2 BioLegend 303204
    CD38 PE-Dazzle594 HIT2 BioLegend 303538/303537
    CD45RB** PE MEM-55** BioLegend 310204
    CD69 BUV395 FN50 BD Biosciences 564364
    CD3 PE-Cy7* various BD Biosciences 563423
    CD7 PE-Cy7* various BioLegend 343114/343113
    CD14 PE-Cy7* various BD Biosciences 557742
    CD16 PE-Cy7* various BioLegend 302015/302016
    CD33 PE-Cy7* various BioLegend 366618/366617
    CD61 PE-Cy7* various BioLegend 336416/336415
    CD235a PE-Cy7* various BioLegend 306619/306620
    CD73 BUV805 AD2 BD Biosciences 748584
    CD95 (FAS) BV711 DX2 BD Biosciences 561633
    HLA-DR BUV737 G46-6 BD Biosciences 748339
    IgA*** PerCp-Vio700 IS11-8E10 Miltenyi 130-113-478
    IgD PE-Cy5 IA6-2 BioLegend 348249/348250
    IgG BUV496 G18-145 BD Biosciences 741172
    IgM BUV563 UCH-B1 BD Biosciences 74892
    Viability FVS510 nan BD Biosciences 564406

    * 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 Name X Feature Y Feature Quantified Cell Type
    Live_cells CD19 Lin  
    └ B_cells_filter1 CD19 CD20  
      ├ plasma_cells_filter1 CD38 CD27  
      ┆└ plasma_cells_filter2     Plasma cell
      └ B_cells_filter2 CD19 CD11c  
           ├ efector_B_cells CD27 CD11c  
           ┆ ├ CD27_neg_effector_B_cells     CD27- effector B cell
           ┆ └ CD27_pos_effector_B_cells     CD27+ effector B cell
           └ not_effector CD32 CD23  
              ├ t2_memory_B_cells     Type 2 polarized memory B cell
              └ not_t2_memory_B_cells CD27 CD45RB  
                ├ antigen_inexperienced_B_cells CD24 CD38  
                ┆  ├ naive_B_cells     Naive B cell
                ┆  └ transitional_B_cells     Transitional B cell
                └ antigen_experienced_B_cells CD27 CD95  
                    ├ CD95_memory_B_cells     CD95 memory B cell
                    └ not_CD95_memory_B_cells CD27 CD45RB  
                        ├ Early_memory_B     Early memory B cell
                        ├ Core_memory_B     Core memory B cell
                        └ Late_memory_B     Late 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

    Feature 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