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Anti cd4 bv605

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Anti-CD4-BV605 is a flow cytometry antibody that binds to the CD4 surface marker on human T cells. It is conjugated to the fluorescent dye Brilliant Violet 605 for detection and quantification of CD4+ cells in samples.

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Lab products found in correlation

Anti cd3 fitc, by BioLegend (4 mentions) Anti cd8 fitc, by BD (4 mentions) Anti cd4 pe, by BioLegend (3 mentions) Anti pd 1 bv711, by BioLegend (2 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Pe cy7 anti slamf7, by BioLegend (2 mentions) Anti cd8 apc, by BioLegend (2 mentions) Anti ifn γ apc, by BioLegend (2 mentions) Anti foxp3 pe, by BD (2 mentions) Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions) Anti cd3 af488, by BioLegend (2 mentions) Anti mhcii efluor450, by Thermo Fisher Scientific (2 mentions) Anti f4 80 apc, by Bio-Rad (2 mentions) Anti cd25 percp cy5, by BioLegend (2 mentions) Anti ly6c af700, by BioLegend (2 mentions) Anti mr pe cy7, by BioLegend (2 mentions) Anti cd11c pe dazzle594, by BioLegend (2 mentions) Anti ly6g bv605, by BioLegend (2 mentions) Perm wash, by BD (2 mentions) Live dead blue, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Anti-CD4 (192 citations) CD4 BV605 (22 citations) BV605-conjugated anti-CD4 (4 citations) BV605 anti-mouse CD4 (4 citations) Anti-human CD4-BV605 antibody (2 citations) Anti-CD4–BV605 (clone RM4-5, (2 citations) Anti-CD4 BV605 GK1.5 (2 citations) Anti-CD4 BV605 (RM4-5, (2 citations) Anti-human CD4 BV605 (2 citations) BV605 rat anti-mouse CD4 (1 citations)

17 protocols using anti cd4 bv605

1

Comprehensive Phenotypic Analysis of Tumor-Specific CD4 T Cells

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Tumor-specific CD4 T clones were stained systematically in PBS, 0.2% bovine serum albumin (BSA), 5 mM EDTA, and 0.2% NaN3 with either (i) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), ECD anti-CD8 (BD), PE-Cy7 anti-SLAMF7 (BioLegend), PerCP-Cy5.5 anti-CD57 (BioLegend), APC (allophycocyanin) anti-CD28 (BioLegend), BV711 anti–PD-1 (BioLegend), PE anti-TCRαβ (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) or (ii) FITC anti-CD3 (BioLegend), BV605 anti-CD4 (BioLegend), APC anti-TRAIL (BioLegend), PE anti-FasL (BioLegend), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) at 4°C for 30 min. Data were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (v.10.4.2).
Cachot A., Bilous M., Liu Y.C., Li X., Saillard M., Cenerenti M., Rockinger G.A., Wyss T., Guillaume P., Schmidt J., Genolet R., Ercolano G., Protti M.P., Reith W., Ioannidou K., de Leval L., Trapani J.A., Coukos G., Harari A., Speiser D.E., Mathis A., Gfeller D., Altug H., Romero P, & Jandus C. (2021). Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer. Science Advances, 7(9), eabe3348.
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2

Multiparameter Flow Cytometry of T and B Cells

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The conjugated monoclonal antibodies used for flow cytometry studies of
T and B cells were: anti-CD45RA APC-CY7, anti-CD56 PE, anti-CD3 FITC, anti-CD4
BV605, anti-CD8 APC and anti-CCR7 Bv421, all from BioLegend.
Alosaimi M., Hoenig M., Jaber F., Platt C.D., Jones J., Wallace J., Debatin K.M., Schulz A., Jacobsen E., Möller P., Shamseldin H.E., Abdulwahab F., Ibrahim N., Alardati H., Almuhizi F., Abosoudah I.F., Basha T.A., Chou J., Alkuraya F.S, & Geha R.S. (2019). Immunodeficiency and Epstein Barr virus induced lymphoproliferation caused by 4-1BB deficiency. The Journal of allergy and clinical immunology, 144(2), 574-583.e5.
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3

Intracellular Cytokine Profiling of T-Cell Response

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The splenocyte-derived cytokines were measured using intracellular cytokine staining (ICS), as described in the Supplementary Materials. Briefly, splenocytes were collected 3 weeks after the third immunization and subsequently pulsed with an anti-CD28 agonist antibody (BD Biosciences, USA) in combination with either RBD or a set of 14-mer peptides covering the RBD region in overlap (NR-52402, BEI Resources, USA). After 1 h, the cells were treated with brefeldin A (BD Biosciences, USA) and incubated for 12 h at 37 °C and 5 % CO2. Non-pulsed cells served as the negative control. Extracellular staining was performed using Live/Dead Fixable Aqua Dead, anti-CD3 BV786 (BioLegend, USA), anti-CD8 APC (BioLegend, USA), and anti-CD4 BV605 (BioLegend, USA). After washing, the fixed and permeabilized cells were intracellularly stained with anti-IFNγ PE (BioLegend, USA), anti-IL-2 BV421 (BioScience), and anti-TNFα Pe-Cy7 (BD Bioscience, USA). The cells were subsequently analyzed using an LSR Fortessa flow cytometer (BD Biosciences, USA).
Rodrigues-Jesus M.J., Teixeira de Pinho Favaro M., Venceslau-Carvalho A.A., de Castro-Amarante M.F., da Silva Almeida B., de Oliveira Silva M., Andreata-Santos R., Gomes Barbosa C., Brito S.C., Freitas-Junior L.H., Boscardin S.B, & de Souza Ferreira L.C. (2022). Nano-multilamellar lipid vesicles promote the induction of SARS-CoV-2 immune responses by a protein-based vaccine formulation. Nanomedicine, 45, 102595.
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4

Multi-Marker Immune Cell Profiling

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Five × 105 unstimulated and PHA (4 μg/mL)
stimulated PBMCs were stained on ice for 30 min with anti-4-1BB PE, anti-4-1BBL
APC, anti-CD4 BV605, anti-CD8 FITC and anti-CD25 BV421, all from BioLegend. For
4-1BBL binding, PBMCs were incubated first with chimeric CD137L:muCD8 fusion
protein (Axxora ANC-503) then washed and incubated with murine anti-CD8 PE.
Alosaimi M., Hoenig M., Jaber F., Platt C.D., Jones J., Wallace J., Debatin K.M., Schulz A., Jacobsen E., Möller P., Shamseldin H.E., Abdulwahab F., Ibrahim N., Alardati H., Almuhizi F., Abosoudah I.F., Basha T.A., Chou J., Alkuraya F.S, & Geha R.S. (2019). Immunodeficiency and Epstein Barr virus induced lymphoproliferation caused by 4-1BB deficiency. The Journal of allergy and clinical immunology, 144(2), 574-583.e5.
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5

Comprehensive Immune Cell Profiling

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All antibody staining was preceded by 15 min of 1:50 FcγR block in FC buffer, on ice. Extracellular antibodies were then added to FC buffer containing FcγR block, and incubated for 45 min on ice. Intracellular staining was accomplished after surface staining using the Foxp3 staining kit (eBioscience). Myeloid subsets were stained with anti-CD1lb-FITC, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4/80-APC (BioRad). To evaluate Tregs, cells were stained with anti-CD3-AF488, anti-CD4-APC, anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-Foxp3-PE (BD Pharmingen). To assess T cell activation, total tumor cells were incubated for 4 hr at 37°C in a 5% CO2 incubator with Protein Transport Inhibitor Cocktail containing brefeldin A and monensin, or with Cell Stimulation Cocktail containing protein transport inhibitors and PMA/ionomycin (eBioscience). Cells were then stained with anti-CD3-AF488, anti-CD4-PE, and anti-CD8-PerCP-Cy5.5 followed by intracellular stain with anti-IFNγ-APC (BioLegend). In addition, 1E5 CD3+ cells were stimulated with 4E4 CD3/CD28 Dynabeads (ThermoFisher) for 3 days, followed by staining using anti-CD3-PerCP-Cy5.5, anti-CD8-BV650, anti-CD4-BV605, anti-IFNγ-APC, anti-TNFα-BV421 (BioLegend), and anti-GranzymeB-PE (eBioscience).
Barberi T., Martin A., Suresh R., Barakat D.J., Harris-Bookman S., Drake C.G., Lim M, & Friedman A.D. (2018). Absence of host NF-κΒ p50 induces murine glioblastoma tumor regression, increases survival, and decreases T cell induction of tumor-associated macrophage M2 polarization. Cancer immunology, immunotherapy : CII, 67(10), 1491-1503.
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6

CFSE-based T cell proliferation assay

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Cryopreserved PBMC were thawed, resuspended in pre-warmed PBS with 0.1% BSA at a final concentration of 106 cells/ml and labelled with 750 nM CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester; Molecular Probes™) for 10 min at 37°C, 5% CO2. The staining was quenched by adding 5 volumes of ice-cold R10 followed by a 5-min incubation on ice. The cells were pelleted, washed and plated in 96-well round-bottom plates at a concentration of 1 x 106 cells/well. The CFSE-labelled cells were then stimulated with 1.5 μg/ml of each peptide in personalized pools or 1 μg/ml SEB (positive control) and R10 (negative control) for 5 days, stained with a dead cell marker (LIVE/DEAD Fixable Aqua stain; Invitrogen) and anti-CD4-BV605 (BioLegend), anti-CD3-ECD (Beckman Coulter) and anti-CD8-Aleva Fluor 700 (eBioscience) mAbs, fixed and acquired on a BD LSR II flow cytometer. Data analysis was performed using FlowJo software (Tree Star Inc.) with gating shown in S1 Fig.
Moyo N., Borthwick N.J., Wee E.G., Capucci S., Crook A., Dorrell L, & Hanke T. (2017). Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens. PLoS ONE, 12(7), e0181382.
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7

Antibody-Mediated Recognition of Env-Expressing Cells

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Infected cells were washed twice in PBS, and aliquoted into 96 well plates (2×105 cells/well for PBMCs, 5×104 for cell lines). Cells were then pelleted and resuspended in PBS containing 25 μg/ml of the appropriate anti-Env antibody, and incubated for 60 minutes at RT. The plate was then washed twice with PBS, and extracellular staining was performed with Anti-CD3-AF700 (Biolegend clone SK7), Anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD clone HIT8a), anti-Fc APC (Biolegend clone HP6017), and Live/Dead Blue (ThermoFisher) and incubated for 20 minutes at RT. Samples were then washed twice and cells were fixed with Fix/Perm solution (BD), washed twice with Perm/Wash (BD) and resuspended in Perm/Wash containing anti-gag KC-57-RD1 (Beckman) and incubated for 20 minutes. Cells were then washed twice with Perm/Wash and resuspended in PBS for data acquisition by flow cytometry. To avoid potential differences in env expression, all HC antibodies and controls were compared for recognition on the same batch of infected cells at the same time. Infected-cell recognition rates among each virus were Z-scored using the standardize function in Microsoft Excel. Negative controls include nonspecific IgG (Biolegend), IVIG, and PBS conditions. Positive controls include polyclonal HIVIG, and combinations of bNAbs directed against multiple binding sites (such as 10E8, PGT121, and 3BNC117).
Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.
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8

Tumor Tissue Characterization by Flow Cytometry

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Mouse tumors were minced and incubated in collagenase from Clostridium histolyticum (Sigma), 2 mg/ml, and DNase I from bovine pancreas, 25 μg/ml (Sigma) in HBSS (Sigma) for 20-30 min at 37°C. The lysate was strained through 70 μm strainers (Fisher Scientific). Cells were counted and 0.5-1x106 cells stained in PBS, 2.5% BSA, 2 mM EDTA after blocking with Trustain (Biolegend) for 15 min. Staining antibodies were diluted 1:200 unless differently specified: anti-CD45-BV785 (1:100, Biolegend), anti-CD3 PE-Cy7 1:50 (Biolegend), anti-CD4 BV605 1:100 (Biolegend), anti-CD8 APC (eBioscience), anti-CD11b BV650 (Biolegend), anti-CD11c FITC (eBioscience), anti-F4/80 PE (Biolegend), anti-Ly6C eFluor450 (eBioscience, 1:100), anti-Ly6G(Gr1) AF700 (eBioscience), anti-CD19 PerCP-Cy5.5 (eBioscience), anti-MHCII APC-Cy7 (Biolegend). Viability was assessed with Fixable Viability Dye eFluor506 (eBioscience) diluted 1:500. Staining was performed for 30 min at 4°C. The cells were finally washed, fixed and analyzed on a BD LSR Fortessa cytometer. Appropriate Fluorescence Minus One (FMO) controls were used in these experiments. Analysis was performed with the FlowJo software. Human omental tissue was processed and stained similarly as previously described (Böhm et al., 2016 (link)).
Maniati E., Berlato C., Gopinathan G., Heath O., Kotantaki P., Lakhani A., McDermott J., Pegrum C., Delaine-Smith R.M., Pearce O.M., Hirani P., Joy J.D., Szabova L., Perets R., Sansom O.J., Drapkin R., Bailey P, & Balkwill F.R. (2020). Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment. Cell Reports, 30(2), 525-540.e7.
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9

HIV-1 ADCC Assay with Activated CD4+ T Cells

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For killing of primary target cells, PBMCs were isolated into buffy coats and split into two aliquots. Target cells were generated by activation with 3 μg/ml of PHA for 3 days, followed by CD4 isolation via negative selection (StemCell Technologies, Inc.) and spinoculation with 0.5 IU/cell of NL4–3 (1,200 × g for 2 hours). After spinoculation, they were washed three times, and cultured for 4 days in R10–50. Before addition of effectors, infected CD4s were CellTrace far red stained (Invitrogen) for discrimination of target cells. Effector PBMCs were cultured with R10–50 with IL-15 added (1 ng/ml), and added a ratio of 5:1 E:T for 18 hours before staining. Staining was carried out using Blue Viability Dye (Invitrogen), anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD Biosciences, clone HIT8a), followed by fixation and permeabilization for p24 staining. ADCC score was calculated as above.
Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.
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10

Antibody-Mediated Recognition of Env-Expressing Cells

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Infected cells were washed twice in PBS, and aliquoted into 96 well plates (2×105 cells/well for PBMCs, 5×104 for cell lines). Cells were then pelleted and resuspended in PBS containing 25 μg/ml of the appropriate anti-Env antibody, and incubated for 60 minutes at RT. The plate was then washed twice with PBS, and extracellular staining was performed with Anti-CD3-AF700 (Biolegend clone SK7), Anti-CD4-BV605 (Biolegend clone OKT4), anti-CD8 FITC (BD clone HIT8a), anti-Fc APC (Biolegend clone HP6017), and Live/Dead Blue (ThermoFisher) and incubated for 20 minutes at RT. Samples were then washed twice and cells were fixed with Fix/Perm solution (BD), washed twice with Perm/Wash (BD) and resuspended in Perm/Wash containing anti-gag KC-57-RD1 (Beckman) and incubated for 20 minutes. Cells were then washed twice with Perm/Wash and resuspended in PBS for data acquisition by flow cytometry. To avoid potential differences in env expression, all HC antibodies and controls were compared for recognition on the same batch of infected cells at the same time. Infected-cell recognition rates among each virus were Z-scored using the standardize function in Microsoft Excel. Negative controls include nonspecific IgG (Biolegend), IVIG, and PBS conditions. Positive controls include polyclonal HIVIG, and combinations of bNAbs directed against multiple binding sites (such as 10E8, PGT121, and 3BNC117).
Rossignol E.D., Dugast A.S., Compere H., Cottrell C.A., Copps J., Lin S., Cizmeci D., Seaman M.S., Ackerman M.E., Ward A.B., Alter G, & Julg B. (2021). Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells. Cell reports, 35(8), 109167.
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