Gl7 pe

Manufactured by BioLegend

GL7-PE is a fluorescently labeled antibody that binds to the GL7 antigen, which is expressed on germinal center B cells. It can be used for the identification and analysis of germinal center B cells in flow cytometry applications.

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Anti-GL7-PE (clone GL7), (2 citations) Anti-GL7 PE (2 citations)

3 protocols using gl7 pe

Multiparameter Analysis of Immune Cell Subsets

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Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).

Tang C.H., Chang S., Paton A.W., Paton J.C., Gabrilovich D.I., Ploegh H.L., Del Valle J.R, & Hu C.C. (2018). Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization. The Journal of Cell Biology, 217(5), 1739-1755.

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Detailed Flow Cytometry for B Cell Subsets

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Cells were resuspended at 10⁶ per 100μl of FACS buffer (1X PBS, 1%BSA, and 2mM EDTA) and blocked with anti-Fc (anti-CD16/CD32) (Tonbo Biosciences, 2.4G2). The following antibodies were used to for staining: B220-PE-Cy7 (Tonbo Biosciences RA3-6B2), CD138-BV711 (BD, 281–2), GL7 eFluor660 (eBioscience, GL-7), CD11b-FITC (Tonbo Biosciences, M1/70), and Ghost Dye^™ Red 780 (Tonbo Biosciences 13–0865) to asses viability. The Annexin V FITC Apoptosis Detection Kit (eBioscience BMS500FI-100) was used to assess cell death. Cells were stained for 40 min and fixed using 1% paraformaldehyde.
Enrichment of CD138+ PB was performed by staining with CD138-APC (BD, 281–2), followed by magnetic enrichment using anti-APC MicroBeads (Miltenyi # 130-090-855). For RNA-seq, GL7+ cells were further enriched from the CD138-depleted fractions using GL7-PE (Biolegend #144608) and anti-PE MicroBeads (Miltenyi #130-105-639).
Flow cytometry was performed on a BD LSRII using FACSDiva (v6.2) and analyzed using FlowJo software. The following gating strategy preceded all flow cytometry analyses presented. Cells were gated on 1) lymphocytes (forward light scatter [FSC]–area by side scatter [SSC]–area), 2) singlets (FSC-height by FSC- width), 3) singlets (SSC- height by SSC-width), 4) live cells (Viability Dye negative), with 5) the exclusion of contaminating macrophages bearing CD11b (Supplemental Figure2A).

Liang F.T, & Philipp M.T. (2000). Epitope Mapping of the Immunodominant Invariable Region of Borrelia burgdorferi VlsE in Three Host Species. Infection and Immunity, 68(4), 2349-2352.

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Multicolor Flow Cytometry Antibody Panel

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Commercial antibodies and staining reagents, from BioLegend: GL7-PacBlue, GL7-PE, GL7-A647, B220-PerCP-Cy5.5, B220-APC-Cy7, IgMb-FITC, IgMa-PE, CD45.2-FITC, CD45.2-APC, CD45.1-FITC, CD45.1-PE, IgD-PB, CD21/35 (7E9)-PE, CD138-PE, CD38-PE-Cy7, CD31-A647, CD157-PE, Streptavidin-PE/Cy7; from eBioscience: CD95 (APO-1/Fas)-PE (clone 15A7) and viability dye Fixable live/dead stain Efluor780; from ThermoFisher Scientific: Rabbit-anti-Goat-A488, Hoechst 33342, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), Phalloidin-A568; from Southern Biotech: AP-Goat-anti-Mouse IgG, AP-Goat-anti-Mouse IgG2a, AP-Goat-anti-Mouse IgG2c; from Rockland Immunochemicals: Rabbit polyclonal anti-B-Phycoerythrin; from DAKO: Rabbit polyconal anti-Mouse Immunoglobulins-biotin; from Perkin-Elmer: Europium-labeled streptavidin. In-house generated anti-idiotypic Ab, clone 9D11, conjugated to Alexa Fluor 647 or Alexa Fluor 568; in-house generated Rabbit polyclonal anti-C3b conjugated to Alexa Fluor 633.

Degn S.E., van der Poel C.E., Firl D.J., Ayoglu B., Al Qureshah F.A., Bajic G., Mesin L., Reynaud C.A., Weill J.C., Utz P.J., Victora G.D, & Carroll M.C. (2017). Clonal Evolution of Autoreactive Germinal Centers. Cell, 170(5), 913-926.e19.

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