Comprehensive B Cell Immunophenotyping

For flow cytometry, we used fluorochrome- or biotin-labeled antibodies against B220 (clone RA3–6B2), CD1d (clone 1B1), CD16/CD46 (Fc block; clone 2.4G2), CD19 (clone 1D3), CD21/CD35 (clone 7G6), CD23 (clone B3B4), CD35 (clone 8C12), CD38 (clone 90), CD95 (clone Jo2), CD138 (clone 281-2), GL-7 (clone GL-7), IgD (clone 11–26c.2a), IgM (clone R6–60.2), IgM^a (clone DS-1), IgM^b (clone AF6-78), and C3 split products (clone RmC11H9); all were purchased from BD Biosciences, Biolegend, eBioscience, or Cedarlane Laboratories. Rabbit polyclonal IgG anti-SRBC was prepared in-house (26 (link)). Staining was done in FACS buffer (PBS 2% FBS; Sigma Aldrich) or, when two or more Brilliant Violet™ antibody conjugates were used, in Brilliant Stain Buffer (BD Biosciences). All antibodies were used at ≤0.5 µg per 1x10⁶ cells.

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Palm A.K., Westin A., Ayranci D, & Heyman B. (2024). Endogenous complement-activating IgM is not required for primary antibody responses but promotes plasma cell differentiation and secondary antibody responses to a large particulate antigen in mice. Frontiers in Immunology, 14, 1323969.

Publication 2023

B220 (clone RA3–6B2), CD19 (clone 1D3), CD23 (clone B3B4), CD95 (clone Jo2), CD138 (clone 281-2), FACS buffer Brilliant Stain Buffer

Corresponding Organization : Uppsala University

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Variable analysis

independent variables

Fluorochrome- or biotin-labeled antibodies against B220 (clone RA3-6B2)
CD1d (clone 1B1)
CD16/CD46 (Fc block; clone 2.4G2)
CD19 (clone 1D3)
CD21/CD35 (clone 7G6)
CD23 (clone B3B4)
CD35 (clone 8C12)
CD38 (clone 90)
CD95 (clone Jo2)
CD138 (clone 281-2)
GL-7 (clone GL-7)
IgD (clone 11-26c.2a)
IgM (clone R6-60.2)
IgM^a (clone DS-1)
IgM^b (clone AF6-78)
C3 split products (clone RmC11H9)

dependent variables

Not explicitly mentioned

control variables

FACS buffer (PBS 2% FBS; Sigma Aldrich)
Brilliant Stain Buffer (BD Biosciences)

controls

Positive control: Rabbit polyclonal IgG anti-SRBC prepared in-house (26 (link))
Negative controls: Not explicitly mentioned

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