Comprehensive B Cell Immunophenotyping

Bone marrow and spleen single cell suspensions were stained for dead cell exclusion using Live/Dead fixable violet dead cell staining kit (Invitrogen Life Technologies, Grand Island, NY). Surface markers were stained with a mixture of fluorochrome-conjugated antibodies, which included: CD19 (clone 6D5, BioLegend, San Diego, CA), B220 (clone RA3-6B2, eBioscience, San Diego, CA), CD138 (clone 281–2, BD bioscience, San Jose, CA), IgD (clone 11.26c.2a, BioLegend, San Diego, CA), IgM (clone II/41, eBioscience, San Diego, CA), IgG (eBioscience, San Diego, CA), MHC class II (clone 500A2, eBioscience, San Diego, CA), CD80 (clone I6-10A1, BD bioscience, San Jose, CA) CD86 (clone GL-1, BioLegend, San Diego, CA).
Fluorescence minus one (FMO) controls were included in each staining protocol and used to set specific gates (22 (link), 26 , 27 (link)). 6-peak validation beads were used for calibration during the time course analysis. Samples were run on a 12-color LSRII cytometer (BD bioscience, San Jose, CA) and analyzed by Flow Jo software (Tree Star Inc., Ashland, OR).

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Ramon S., Baker S.F., Sahler J.M., Kim N., Feldsott E.A., Serhan C.N., Martínez-Sobrido L., Topham D.J, & Phipps R.P. (2014). The specialized proresolving mediator 17-HDHA enhances the antibody-mediated immune response against influenza virus: Anew class of adjuvant?. Journal of immunology (Baltimore, Md. : 1950), 193(12), 6031-6040.

Publication 2014

Live/Dead fixable violet dead cell staining kit CD19 (clone 6D5, B220 (clone RA3-6B2, CD138 (clone 281–2, IgM (clone II/41, CD86 (clone GL-1, 12-color LSRII cytometer

Antibodies Bone marrow Cd138 Cell Clone Fluorescence Fluorochrome Mhc class ii Spleen Tree Violet

Corresponding Organization : University of Rochester

Other organizations : Brigham and Women's Hospital, Harvard University

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Variable analysis

independent variables

Bone marrow and spleen single cell suspensions

dependent variables

Expression of CD19, B220, CD138, IgD, IgM, IgG, MHC class II, CD80, CD86

control variables

Live/Dead fixable violet dead cell staining
Fluorescence minus one (FMO) controls to set specific gates
6-peak validation beads for calibration during the time course analysis

controls

Positive controls: FMO controls
Negative controls: Not explicitly mentioned

Annotations

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