Cxcr5 bv421

Manufactured by BD

CXCR5-BV421 is a flow cytometry antibody that binds to the CXCR5 receptor. CXCR5 is a chemokine receptor that plays a role in the homing and migration of B cells. The CXCR5-BV421 antibody is conjugated with the BV421 fluorescent dye, allowing for the detection and analysis of CXCR5-expressing cells using flow cytometry.

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5 protocols using cxcr5 bv421

Multiparameter Immune Cell Profiling

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PBMCs were stained with the following antibodies: CD3-APC/Fire750 (BioLegend), CD14-APC/Fire750 (BioLegend), CD20-APC/Fire750 (BioLegend), CD56-APC (Tongsheng Shidai) and CXCR5-BV421 (BD Biosciences). Data were acquired using an Amnis ImageStreamX Mark II Imaging Flow Cytometer (Amnis, Seattle, WA, USA) and analyzed using the Amnis IDEAS software v6.2 (Amnis).

Guo A.L., Jiao Y.M., Zhao Q.W., Huang H.H., Deng J.N., Zhang C., Fan X., Xu R.N., Zhang J.Y., Zhen C., Xie Z.M., Qin Y.M., Xu J.Q., Yang Y., Shi M., Huang L., Song J.W, & Wang F.S. (2021). Implications of the accumulation of CXCR5+ NK cells in lymph nodes of HIV-1 infected patients. EBioMedicine, 75, 103794.

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Multiparametric Flow Cytometric Immunophenotyping

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Peripheral blood mononuclear cells were washed in PBS with 1% BSA (bovine serum albumin) and incubated for 30 min at 25°C with fluorescently labeled antibodies specific for B and T cells. Subsequently, samples were centrifuged and resuspended in propidium iodide (PI) solution (1 μg/ml PI and 10 μg/ml RNase A in PBS) and analyzed using BD FACS Canto II flow cytometer (BD Biosciences). The B cell panel included the following antibodies: IgA FITC, IgD PE, CD3 PerCP-Cy5.5, CD27 APC-H7, CD19 PE-Cy7, β7 APC, and CD38 BV421 from BD Biosciences. The T cell panel included the following antibodies: CXCR5 BV421, CXCR3 PE, CD4 APC-H7, CD3 FITC, CD196 APC, CD279 (PD-1) BV510, and CD45RA PE-Cy7 from BD Biosciences. Results were analyzed using FACSDiva software (BD Biosciences) and reported as MFI, reflecting the levels of cell surface antigens and relative cell count with respect to hierarchically higher cell populations (%). Cellular aggregates were eliminated using morphology parameters (FSC-A and FSC-H) (see Supplementary Figures 1, 2 for gating strategy).

Lodde V., Floris M., Beerman I., Munk R., Guha R., Steri M., Orrù V., Abdelmohsen K., Crompton P.D., Gorospe M., Idda M.L, & Cucca F. (2020). Evolutionarily Selected Overexpression of the Cytokine BAFF Enhances Mucosal Immune Response Against P. falciparum. Frontiers in Immunology, 11, 575103.

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Comprehensive Immune Cell Profiling

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Fluorochrome-conjugated anti-mouse monoclonal antibodies CD3-AF700 (cat # 100216), CD4-BV785 (cat # 100453), CD8-PB (cat # 100725), CD25-BV650 (cat # 102038), GITR-PECy7 (cat # 120222), ICOS-PECy5 (cat # 107708), IFNg-PeCy7 (cat # 505826), and IL-2-PB (cat # 503820) were purchased from Biolegend. CD44-Percp-cyanine5.5 cat# 45-0441-80, CD62L-APCeFL780 (cat # 47-0621-82), Foxp3-APC (cat# 17-5773-82), Eos-eFL660 (cat # 50-5758-80), Helios-PeCy7 (cat # 25-9883-42), Aiolos-PE (cat # 12-5789-80),and bCatenin-eFL660 (cat # 50-2567-42) were purchased from Thermo Fisher Scientific. PD-1-PECy7 (cat# 25-9985-80), CXCR5-BV421 (cat # 562889), Bcl-6-PE (cat # 569522), and phospho-STAT5-PE (pY694) (cat # 612567) were procured from BD Biosciences.

Thomas R.M., Pahl M.C., Wang L., Grant S.F., Hancock W.W, & Wells A.D. (2024). Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function. eLife, 12, RP91392.

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Multiparameter Flow Cytometry of GC B Cells

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One million cells were stained in PBS+1% FBS. Cocktails of antibodies against the following surface proteins were used: B220 PerCP-Cy5.5 (RA3-6B2), B220 FITC (RA3-6B2), GL7 BV421 (GL7), IgD BV786 (11-26c.2a), Ly6G PE (1A8), CD11b PerCP-Cy5.5 (M1/70), CD11b PE-Cy7 (M1/70), CD4 APC (RM4-5), CxCR5 BV421 (2G8), CD11c BV421 (HL3) (All BD) CD38 PE-Cy7 (90), F4/80 PE-Cy7 (BM8), F4/80 APC-EF780 (eBioscience), CD11c APC-Cy7 (N418), and PD-1 BV605 (29F.1A12) (Biolegend). A live/dead marker was used to exclude dead cells in the GC B and TFH cell panels (Fixable Viability Dye eFluor™ 780, eBioscience). AF647-labeled ovalbumin (OVA-AF647) was from Invitrogen. Antigen-specific germinal center B cells were measured by including CTH522 coupled to AF488 as probe (conjugated by Life technologies at a coupling ratio of 3 moles dye/mole). Cells were analyzed on a BD Fortessa or FACSCanto flow cytometer.

Pedersen G.K., Wørzner K., Andersen P, & Christensen D. (2020). Vaccine Adjuvants Differentially Affect Kinetics of Antibody and Germinal Center Responses. Frontiers in Immunology, 11, 579761.

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Multiparametric Phenotyping of Germinal Center Cells

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Cells from the inguinal lymph nodes were collected from all individuals; the cells were plated (10⁶ cells/well) and treated with Mouse BD Fc Block™ (BD Pharmingen, 553142) to block non-antigen-specific bindings of immunogloblulins to Fcγ III and Fcγ II receptors. Two panels of antibodies were used to stain cell populations in the germinal centers (GC) of the lymph nodes; GC B cells (B220⁺ IgD⁻ CD38⁻ GL7⁺) and T follicular helper cells (TFH) (B220⁻ CD4⁺ PD-1⁺ CXCR5⁺). The following antibodies were mixed in PBS 1% FBS: GL7-FITC (BioLegend GmbH, Koblenz, Germany; 144604), IgG1-PE (BD Pharmingen, 550083), B220-PerCP-Cy5.5 (BD Pharmingen, 552771), CD38-PE-CY7 (eBioscience, 25-0381) and IgD-BV786 (BD Pharmingen, 563618) to stain the B cells in the GC. To stain the TFH cell population, the following antibody panel was used: CD4-FITC (BD Pharmingen, 553047); CD279(PD1)-PE (BD Pharmingen, 551892); CD45R(B220)-PerCP-Cy5.5 (eBioscience, 65-0865-14), CXCR5-BV421 (BD Pharmingen, 562889) and live/dead-EF780 (BD Pharmingen, 562889). After an incubation period of 30 min at 4 °C, the cells were acquired on the FACS LSRII instrument using DIVA software (BD Bioscience, USA) and all the acquired data analyzed using FlowJo software (Tree Star Inc.). Compensation beads (BD Pharmingen, USA) were used to compensate the fluorophores.

Sisteré-Oró M., Pedersen G.K., Córdoba L., López-Serrano S., Christensen D, & Darji A. (2020). Influenza NG-34 T cell conserved epitope adjuvanted with CAF01 as a possible influenza vaccine candidate. Veterinary Research, 51, 57.

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