Anti mouse cd4 bv510

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Anti-mouse CD4-BV510 is a fluorochrome-conjugated monoclonal antibody that binds to the CD4 protein expressed on the surface of mouse T helper cells.

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Anti-CD4 (192 citations) Anti-mouse CD4 (38 citations) CD4-BV510 (20 citations) Anti-CD4-BV510 (14 citations)

8 protocols using anti mouse cd4 bv510

Characterization of Murine Lymphocyte Subsets

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IELs and LPLs were passed through a 40-um cell strainer to a obtain single-cell suspension. A single-cell suspension was stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) and incubated with brefeldin A (5 ug/ml) for 5 h, followed by staining for intracellular cytokines and surface markers. Exclusion of dead cells was performed with LIVE/DEAD Fixable Zombie Dead Cell Stain Kit (BioLegend). All cell preparations were Fc-blocked by CD16/32 antibody (BioLegend) prior to staining. Cell surface staining was performed with PerCP/Cy5.5 anti-mouse CD8α, FITC anti-mouse CD3, BV510 anti-mouse CD4, APC anti-mouse CD103, PE anti-mouse CD69 antibody and BV421 anti-mouse CD62L antibody (all from BioLegend). For detection of intracellular cytokines, cells were fixed in 4% PFA and permeabilized with BD perm/wash™ (BD Biosciences), followed by staining with Bv421 anti-mouse TNF-αand AF647 anti- mouse IFN-γ (BioLegend). Flow cytometric analysis was performed on an LSR Fortessa, and cell results were acquired using Diva software (BD Biosciences) and analyzed with FlowJo software. Sorting was performed on an Aria SORP high-speed cell sorter (BD Biosciences).

Shi F., Zhang S., Zhang N., Yu Y., Sun P., Tang X., Liu X, & Suo X. (2023). Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice. Frontiers in Immunology, 14, 1128637.

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Comprehensive Multiparameter Immune Profiling

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Alkaline Phosphatase (AP) anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG2c and IgG3 were acquired from Southern Biotech, while AP anti-mouse IgG was purchased from Bio-rad. BV421 anti-mouse I-A^b, PE anti-mouse I-A^d, BV421 anti-mouse H-2K^b, BV421 anti-mouse GL7, BV480 anti-mouse IgM, BV510 anti-mouse CD38, BB515 anti-mouse CD19, BV650 anti-mouse RORγt, PE CF594 anti-mouse GATA3, BB515 anti-mouse CD8α, and Alexa Fluor 647 (AF647) anti-mouse Bcl6 were obtained from BD Biosciences. FITC anti-mouse I-A^k, AF700 anti-mouse CD45, BV510 anti-mouse H-2K^d, PE anti-mouse H-2K^k, BV605 anti-mouse CD19, AF700 anti-mouse T cell receptor (TCR), AF700 anti-mouse CD11b, AF700 anti-mouse CD11c, BV650 anti-mouse IgD, BV421 anti-mouse CD3, BV510 anti-mouse CD4, BV605 anti-mouse Tbet, BV785 anti-mouse CD25, PerCP Cy5.5 anti-mouse CD19, PerCP Cy5.5 anti-mouse CD11c, PerCP Cy5.5 anti-mouse CD11b, and AF700 anti-mouse CD44 were purchased from Biolegend.

Patel S.R., Lundgren T.S., Baldwin W.H., Cox C., Parker E.T., Healey J.F., Jajosky R.P., Zerra P.E., Josephson C.D., Doering C.B., Stowell S.R, & Meeks S.L. (2022). Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway. Frontiers in Immunology, 13, 880829.

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Characterizing PD-1 Knockout EL4 Cells

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Female C57BL/6 mice were purchased from The Jackson Laboratories. Animal studies were conducted following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah. EL4 (ATCC^® TIB-39^™) cells were purchased from ATCC and were maintained in DMEM medium with 10% horse serum. EL4 (PD-1^KO) cells were generated according to our previous research and maintained in the same medium for EL4 [15 (link)]. Macrophage Raw 264.7 cells were purchased from ATCC and maintained in RPMI 1640 medium with 10% FBS. Expi293 expression system was purchased from ThermoFisher and applied following the manufacture instruction. PE anti-mouse IgG2a antibody (clone: m2a-15F8) was purchased from Ebioscience. APC anti-mouse CD3 (clone: 17A2), FITC anti-mouse CD3 (clone:17A2), BV510 anti-mouse CD4 (clone: RM4–5), APC/Cyanine7 anti-mouse CD8 (clone: 53–6.7), PE anti-mouse TCR Vβ12 (clone: MR11–1), APC anti-mouse PD-1 (homemade), APC anti-mouse CD11b (clone: M1/70), PE anti-mouse PD-1 (clone: RMP1–14) and PE anti-mouse FcγRIV (clone: 9E9) were purchased from Biolegend.

Rock C.O., Heath R.J., Park H.W, & Jackowski S. (2001). The licC Gene of Streptococcus pneumoniae Encodes a CTP:Phosphocholine Cytidylyltransferase. Journal of Bacteriology, 183(16), 4927-4931.

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Splenocyte Stimulation and Flow Cytometry

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Splenocytes were processed as described in the previous section and stimulated with RBD peptides for 5 hours at 37°C with protein transport inhibitor (Invitrogen) and anti-mouse CD107a-FITC antibody (BioLegend). Cell stimulation cocktail and R10, with protein transport inhibitor, were used as positive and negative controls, respectively. After stimulation, cells were stained with Live/Dead violet (Invitrogen) for viability. Anti-mouse CD4-BV510, CD8-APC-Cy7, CD44-A700, and CD62L-BV711 antibodies were used for surface staining and CD3e-PE-Cy5, IFN-γ-APC, and TNF-α-BV605 (all from BioLegend) were used for intracellular staining. The samples were run on an 18-color LSRII flow cytometer (BD Biosciences) and analyzed by FlowJo software.

Konrath K.M., Liaw K., Wu Y., Zhu X., Walker S.N., Xu Z., Schultheis K., Chokkalingam N., Chawla H., Du J., Tursi N.J., Moore A., Adolf-Bryfogle J., Purwar M., Reuschel E.L., Frase D., Sullivan M., Fry B., Maricic I., Andrade V.M., Iffland C., Crispin M., Broderick K.E., Humeau L.M., Patel A., Smith T.R., Pallesen J., Weiner D.B, & Kulp D.W. (2022). Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection. Cell Reports, 38(5), 110318.

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Epitope-specific T-cell Response Analysis

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Single-cell suspension from spleens of immunized animals (BALB/c mice 35 d.p.i or C57BL/6 mice 28 d.p.i) were prepared as described before and stimulated with 5 μg/mL of peptide pools that span both the lumazine synthase, GT8 or HA domains, or individual Trp2 (SVYDFFVWL) and Gp100₂₅ peptide (EGPRNQDWL) (GenScript) for 5 hours at 37°C in the presence of 1:500 protein transport inhibitor (ThermoFisher) and anti-mouse CD107a-FITC (ThermoFisher). The cells were then incubated with live/dead Fixable Violet Dead Cell Stain Kit (for 405 nm excitation) for 10 minutes at room temperature, and surface stained (anti-mouse CD4-BV510, Biolegend, Catalog: 100559; anti-mouse CD8–APC-Cy7, Biolegend, Catalog: 100714) at room temperature for 30 minutes. The cells were then fixed and permeabilized according to manufacturer’s instructions for BD Cytoperm Cytofix kit and stained with anti-mouse IL2–PE-Cy7 (BioLegend, Catalog: 503832), anti-mouse IFNγ-APC (BioLegend, Catalog: 505810), anti-mouse CD3e–PE-Cy5 (BioLegend, Catalog: 100310), and anti-mouse TNFα-BV605 (BioLegend, Catalog: 506329) at 4°C for 1 hour. The cells were subsequently analyzed with LSR II 18-color flow cytometer. The data was analyzed with FlowJo V10.6.1. ICS gate for CD8⁺IFNγ⁺ population is set as in Figure 1C and Supplementary Figure S4.

Xu Z., Chokkalingam N., Tello-Ruiz E., Wise M.C., Bah M.A., Walker S., Tursi N.J., Fisher P.D., Schultheis K., Broderick K.E., Humeau L., Kulp D.W, & Weiner D.B. (2020). A DNA-launched nanoparticle vaccine elicits CD8+ T-cell immunity to promote in vivo tumor control. Cancer immunology research, 8(11), 1354-1364.

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Murine Splenocyte Activation Assay

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Single-cell suspension from spleens of immunized C57BL/6 mice were prepared as described before and stimulated with peptides that either span the lumazine synthase domain, or individual Trp2₁₈₈ (SVYDFFVWL), Gp100₂₅ (EGPRNQDWL) and Tyrp1₄₅₅ (CTAPDNLGYM) peptides at 5 µg/mL for 5 hours at 37°C in the presence of 1:500 protein transport inhibitor (ThermoFisher) and anti-mouse CD107a-FITC (ThermoFisher). The cells were then incubated with live/dead Fixable Violet Dead Cell Stain Kit (for 405 nm excitation) for 10 minutes at room temperature, and surface stained (anti-mouse CD4-BV510, Biolegend, Catalog: 100559; anti-mouse CD8–APC-Cy7, Biolegend, Catalog: 100714) at room temperature for 30 minutes. The cells were then fixed and permeabilized according to manufacturer’s instructions for BD Cytoperm Cytofix kit and stained with anti-mouse IL2–PE-Cy7 (BioLegend, Catalog: 503832), anti-mouse IFNγ-APC (BioLegend, Catalog: 505810), anti-mouse CD3e–PE-Cy5 (BioLegend, Catalog: 100310), and anti-mouse TNFα-BV605 (BioLegend, Catalog: 506329) at 4°C for 1 hour. The cells were subsequently analyzed with LSR II 18-color flow cytometer. The data was analyzed with FlowJo V10.6.1.

Tursi N.J., Xu Z., Helble M., Walker S., Liaw K., Chokkalingam N., Kannan T., Wu Y., Tello-Ruiz E., Park D.H., Zhu X., Wise M.C., Smith T.R., Majumdar S., Kossenkov A., Kulp D.W, & Weiner D.B. (2023). Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo. Frontiers in Immunology, 14, 1072810.

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Multiparametric Flow Cytometry of Lymphoid Cells

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Single-cell suspension was generated from the spleen as described in the prior section, and from the lymph nodes by applying pressure on the tissues to pass through 40 um strainers. Single-cell suspensions were washed once with PBS, and then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted 333-fold in PBS at room temperature for 10 min, followed by another wash in PBS. The cells were then incubated with mouse Fc-block (anti-mouse CD16/32, BioLegend) in 1% FBS/PBS at room temperature for an additional 5 min, followed by incubation with antibody mixtures (anti-mouse GL7-FITC, anti-mouse CD4 BV510, anti-mouse CD44 AF700, anti-mouse PD1 PE-Cy7, and anti-mouse CXCR5 biotin, BioLegend, each diluted 1:200 in 1% FBS/PBS) without washing for an additional 40 min at room temperature. The cells were washed and then incubated with Streptavidin-APC at 0.5ug/mL in 1% FBS/PBS for 20 min at room temperature. The cells were then washed again and resuspended in 1% FBS/PBS for flow analysis with an LSRII 18-color instrument.

Xu Z., Walker S., Wise M.C., Chokkalingam N., Purwar M., Moore A., Tello-Ruiz E., Wu Y., Majumdar S., Konrath K.M., Kulkarni A., Tursi N.J., Zaidi F.I., Reuschel E.L., Patel I., Obeirne A., Du J., Schultheis K., Gites L., Smith T., Mendoza J., Broderick K.E., Humeau L., Pallesen J., Weiner D.B, & Kulp D.W. (2022). Induction of tier-2 neutralizing antibodies in mice with a DNA-encoded HIV envelope native like trimer. Nature Communications, 13, 695.

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Multiparametric Flow Cytometry Analysis

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Single cell suspension was generated from the spleen as described in the prior section, and from the lymph nodes by applying pressure on the tissues to pass through 40 um strainers. Single cell suspensions were washed once with PBS, and then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted 333-fold in PBS at room temperature for 10 minutes, followed by another wash in PBS. The cells were then incubated with mouse Fc-block (anti-mouse CD16/32, BioLegend) in 1% FBS/PBS at room temperature for additional 5 minutes, followed by incubation with antibody mixtures (anti-mouse GL7-FITC, anti-mouse CD4 BV510, anti-mouse CD44 AF700, anti-mouse PD1 PE-Cy7 and anti-mouse CXCR5 biotin, BioLegend, each diluted 1:200 in 1% FBS/PBS) without washing for an additional 40 minutes at room temperature. The cells were washed and then incubated with Streptavidin-APC at 0.5ug/mL in 1% FBS/PBS for 20 minutes at room temperature. The cells were then washed again and resuspended in 1% FBS/PBS for flow analysis with an LSRII 18-color instrument.

Xu Z., Walker S.N., Wise M.C., Chokkalingam N., Purwar M., Moore A., Tello‐Ruiz E., Wu Y., Majumdar S., Zaidi F.I., Reuschel E.L., Patel I., Obeirne A., Du J., Schultheis K., Gites L., Smith T.R., Mendoza J., Broderick K.E., Humeau L., Pallesen J., Weiner D.B., & Kulp D.W. (2021). Structural identification of neutralizing antibodies induced by nucleic acid encoded native-like trimer.

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