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    B cell Stimulation Flow Cytometry

    We used flow cytometry to measure activation of B cells in at-risk individuals (ARI) and healthy controls. B cells were treated with CpG and CD40L or without stimulation for 48 hours. 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 Fluorophore Clone Vendor Cat. #
    CD3** PE-Cy7 UCHT1 BD Biosciences 563423
    CD7** PE-Cy7 CD7-6B7 BioLegend 343113/343114
    CD10 BV750 HI10a BD Biosciences 747517
    CD11c FITC Bu15 BioLegend 337214
    CD14* BUV395 M5E2 BD Biosciences 740286
    CD14** PE-Cy7 M5E2 BC Biosciences 557742
    CD20 APC-Cy7 2H7 Biolegend 302314
    CD21 BUV661 1048 BD Biosciences 75018
    CD24 PE-Cy5 ML5 BioLegend 311147
    CD27 BV786 L128 BD Biosciences 742731
    CD33** PE-Cy7 P67.6 BioLegend 366617/366618
    CD45RB APC MEM-55 Bio-Rad MCA1114
    CD56** PE-Cy7 B159 BD Biosciences 557747
    CD61** PE-Cy7 VI-PL2 BioLegend 336415/336416
    CD69 BUV737 FN50 BD Biosciences 564364
    CD73 BUV805 AD2 BD Biosciences 748584
    CD95 BV605 DX2 BioLegend 305628
    CD107a PE-Dazzle594 H43A BioLegend 328646
    CD235a** PE-Cy7 HIR2 BioLegend 306619/306620
    HLA-DR BV711 L243 BioLegend 307643
    IgA PerCP-Vio700 IS11-8E10 Miltenyi 130-113-478
    IgD BV480 IA6-2 BD Biosciences 566138
    IgG BUV496 G18-145 BD Biosciences 741172
    IgM BUV563 UCH-B1 BD Biosciences 748929
    IL-10 BV650 JES3-9D7 BD Biosciences 564051
    IL-6 BV421 MQ2-13A5 BD Biosciences 563279
    RANKL PE MIH24 BioLegend 347504
    TNFa Alexa Fluor 700 Mab11 BioLegend 502928
    Viability FVS510 nan BD Biosciences 564406

    *Included in 1 of 3 batches; Not included in final analyses. 
    ** All PE-Cy7 antibodies are included in the "Lin" channel.

    Data from these flow cytometry experiments are provided in two formats.

    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.
    2. A compressed table (.csv.gz) file containing annotated cell data with scaled expression for each measured marker.

    The columns provided in the metadata table and annotated CSV file are provided below:

    Common columns:
    These columns are available in both the .fcs metadata and annotated cell .csv files.

    flow.fileName: The file name of the .fcs file
    flow.batch: The batch of experimental samples - data was generated in 3 batches of experiments.
    flow.condition: The experimental condition (CpG_CD40_stimulated or unstimulated)

    subject.diseaseGroup: Which group each subject belonged to: Control (HC1); Control (HC2); ACPA+ At-risk (ARI); or ACPA+ Early RA (ERA)
    subject.progressionGroup: For ARI subjects, whether subject was diagnosed with RA during the study period (converted): Progression (CONV) or No Progression (NONC)
    subject.subjectID: An ID for the Subject
    subject.biologicalSex: The biological sex of the Subject
    subject.diagnosisAge: For ARI-CONV subjects, age at diagnosis of RA
    subject.diagnosisBMI: For ARI-CONV subjects, Body Mass Index at diagnosis of RA
    subject.biologicalSex: The biological sex of the Subject
    am.bmi: Body Mass Index (BMI)
    infl.anti_ccp3: Anti-CCP3
    infl.rf_iga_result: RFIgA Result
    infl.rf_igm_result: RFIgM Result
    sample.daysToDiagnosis: Days between sample collection and diagnosis of RA for CONV group
    sample.daysSinceFirstVisit: Days between initial sample collection and this sample draw

    Annotation columns:
    These columns are available only in the annotated cell .csv file.

    flow.cellType: The B cell type assigned to the cell.
    flow.isotype: The isotype of the B cell based on Ig detection (if available)
    TNFa_pos: A boolean value (True or False) for TNFa expression.
    IL_6_pos: A boolean value (True or False) for IL-6 expression.
    IL_10_pos: A boolean value (True or False) for IL-10 expression.
    RANKL_pos: A boolean value (True or False) for RANKL expression.

    Feature columns:
    These columns provide scaled expression values for each feature, and are only present in the annotated cell .csv file. See above for additional antibody and fluorophore information.

    CD45RB
    CD20
    TNFa
    IgG
    IgM
    CD21
    CD69
    CD73
    IL_6
    IgD
    CD95
    IL_10
    HLA_DR
    CD10
    CD27
    CD11c
    Viability
    RANKL
    CD24
    Lin
    CD107a
    IgA

    ALTRA B cell flow cytometry
    File Name Description Download Link
    altra_bcell_clustered_scaled_data.csv.gz Annotated cell dataset .csv
    altra_bcell_fcs.tar FCS files and file metadata