Cd4 bv785

Manufactured by BioLegend

Sourced in United States

The CD4-BV785 product is a fluorescently labeled antibody that binds to the CD4 receptor on the surface of T helper cells. It is designed for use in flow cytometry applications to identify and quantify CD4+ T cells in biological samples.

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Lab products found in correlation

Close spelling variants of this product

CD4 BV785 (OKT4; (6 citations) CD4-BV785 (GK1.5) (2 citations) Anti-CD4 BV785 (OKT4, (2 citations) Anti-CD4-BV785 (2 citations) CD4-BV785 (clone OKT4) (2 citations) CD4-BV785 (RM4-5; (2 citations)

17 protocols using cd4 bv785

Flow Cytometric Analysis of Muscle Inflammation

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For flow cytometric muscle analysis, mice were euthanized by CO2 asphyxiation 7 days after injury to analyze cellular inflammation in the muscle. Tissue was minced, digested in 1mg/mL collagenase IA for 45 min at 37°C, filtered through membranes with 40μm pore size, and resuspended in 3% FBS for immunostaining. Cell suspensions were immunostained for 30 min on ice followed by fixation in 2% PFA for 10 min and addition of CountBrightTM Absolute Counting Beads. The following antibody panel was used: MerTK-PE (clone 108928; R&D Systems), CCR7-PE/Cy7 (clone 4B12; BioLegend), CD3-FITC (17A2; BioLegend), CD25-PerCP/Cy5.5 (clone PC61; BioLegend), Ly6C-APC (clone HK1.4, BioLegend), Ly6G-APC/Cy7 (clone 1A8; BioLegend), CD11c-BV421 (clone N418; BioLegend), CD11b-BV510 (clone M1/70; BioLegend), CD206-BV605 (clone C068C2; BioLegend), CD64-BV711 (clone X54-5/7.1; BioLegend), and CD4-BV785 (clone GK1.5; BioLegend). Samples were run on a BD FACS Aria IIIu cytometer and data was analyzed using FlowJo software. Cells were immunophenotyped according to the following gating scheme: macrophage, MerTK+CD64+; dendritic cell, NOT(MerTK+CD64+)CD11c+; monocyte, NOT(MerTK+CD64+)CD11c-CD11b+SSClo; neutrophil, Ly6G+SSChi; T lymphocyte, CD3+; helper T lymphocyte, CD3+CD4+; regulatory T lymphocyte (Treg), CD3+CD4+CD25+.

Krieger J.R., Sok M.C., Turner T.C, & Botchwey E.A. (2018). Delivery of Immunomodulatory Microparticles in a Murine Model of Rotator Cuff Tear. MRS advances, 3(24), 1341-1346.

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Multicolor Cytokine Analysis of Mtb300-specific CD4 T cells

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Cryopreserved cells were thawed, washed and permeabilised with a Transcription Factor perm/wash buffer (eBioscience). Cells were then stained at room temperature for 45 min with the following antibodies: CD3 BV650 (OKT3; BioLegend, San Diego, CA, USA), CD4 BV785 (OKT4; BioLegend), CD8 BV510 (RPA‐T8; BioLegend), CD27 PE‐Cy5 (1A4CD27; Beckman Coulter, Brea), HLA‐DR BV605 (L243; BioLegend), Killer cell Lectin‐like Receptor G1 (KLRG1) PerCP‐eFluor 710 (13F12F2; eBioscience), IFN‐γ BV711 (4S.B3; BioLegend), TNF‐α eFluor 450 (Mab11; BioLegend eBioscience), IL‐2 PE/Dazzle (MQ1‐17H12, BioLegend eBioscience) MIP‐1β Alexa Fluor 488 (#24006; R&D systems, Minneapolis, MN, USA) and CD153 (R&D116614; R&D systems). Samples were acquired on a BD LSR‐II and analysed using FlowJo (v9.9.6, FlowJo LCC, Ashland, OR, USA). A positive cytokine response was defined as at least twice the background of unstimulated cells. To define the phenotype of Mtb300‐specific CD4 T cells, a cut‐off of 30 events was used.

Riou C., Du Bruyn E., Ruzive S., Goliath R.T., Lindestam Arlehamn C.S., Sette A., Sher A., Barber D.L, & Wilkinson R.J. (2020). Disease extent and anti‐tubercular treatment response correlates with Mycobacterium tuberculosis‐specific CD4 T‐cell phenotype regardless of HIV‐1 status. Clinical & Translational Immunology, 9(9), e1176.

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T Cell Phenotyping for SARS-CoV-2 Immunity

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For T cell analysis, single cell suspensions were incubated with FcγR antibody (clone 93, BioLegend) to block non-specific antibody binding, followed by staining with a cocktail of labeled mAbs including Fixable Viability dye eFluor506, CD3e-BV711 (1:100, clone:145–2C11, BD Biosciences), CD8α-PerCP/Cyanine 5.5 (1:100, 53–6.7, BioLegend), CD4-BV785 (1:100, clone: RM4–5, BioLegend), CD44-PE/Cyanine 7 (1:100, clone: IM7, BioLegend), CD69-FITC (1:100, clone: H1.2F3, BioLegend), CD103-PE (1:100, clone: 2E7, BioLegend) and APC-labeled SARS-CoV-2 S- specific tetramer (MHC class I tetramer, residues 539–546, VNFNFNGL, H-2K(B) for 60 min at room temperature. Cells were washed twice with FACS buffer, fixed with 2% paraformaldehyde (PFA) for 20 min prior to data acquisition. Data were acquired on an Aurora (Cytek) spectral flow cytometer and analyzed in FlowJo v10 software.

Ying B., Darling T.L., Desai P., Liang C.Y., Dmitriev I.P., Soudani N., Bricker T., Kashentseva E.A., Harastani H., Schmidt A.G., Curiel D.T., Boon A.C, & Diamond M.S. (2023). A bivalent ChAd nasal vaccine protects against SARS-CoV-2 BQ.1.1 and XBB.1.5 infection and disease in mice and hamsters. bioRxiv.

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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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Immune Cell Phenotyping by Flow Cytometry

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Surface staining was done by incubating cells with different combinations of anti-human CD3-BV 650, CD4-BV 785, CD19-BV 605, Tim-1-APC monoclonal antibodies (BioLegend), and Violet Dead Cell stain (Invitrogen) for 30 min at 4°C. Cells were then washed and treated with Intracellular Fixation and Permeabilization Buffer set (eBioscience). Anti-human IL-10-PE, granzyme A-FITC, granzyme B-APC and/or perforin-PE monoclonal antibodies were then added, followed by 30 min 4°C incubation, after which cells were washed twice and fixed with 2% formaldehyde. Samples were acquired in BD Fortessa and analyzed in FlowJo.

Xue H., Lin F., Tan H., Zhu Z.Q., Zhang Z.Y, & Zhao L. (2016). Overrepresentation of IL-10-Expressing B Cells Suppresses Cytotoxic CD4+ T Cell Activity in HBV-Induced Hepatocellular Carcinoma. PLoS ONE, 11(5), e0154815.

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Isolation and Sorting of CD4 Memory Subsets

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Peripheral blood cells were obtained by leukapheresis. Following lysis of red blood cells, they were preserved in freezing medium at -80C/LN. 3.0 × 10⁸ PBMC (93.1% viability) were thawed and stained with LIVE/DEAD Aqua stain (Molecular Probes, L34957, 5 min, 22°C), CD3-APC-H7 (BD Biosciences, 641406), CD4-BV785 (BioLegend, 317442), CD8-QDot655 (Invitrogen, Q10055), CD11c-PE (BD Biosciences, 347637), CD14-PE (BD Biosciences, 555398), CD27-PE/Cy5 (Beckman Coulter, 6607107), CD45RO-ECD (Beckman Coulter, IM2712U), CD56-APC (BioLegend, 304610), CD57-BV421 (VRC Ab), CCR7-Ax700 (VRC Ab), and TCR γδ (BD Biosciences, 555718) for 15 min at 22°C. Cells were washed, kept on ice, and sorted on a BD FACSAria into CD4 memory subsets as follows: Naïve (CD3+ Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27 + CD45RO-CCR7 + CD57-), Central/Transitional Memory (CTM, CD3+ Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27+ CD45RO+), and Effector Memory (EM, CD3 + Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27-) (Supplementary Figure S1).

Musick A., Spindler J., Boritz E., Pérez L., Crespo-Vélez D., Patro S.C., Sobolewski M.D., Bale M.J., Reid C., Keele B.F., Capoferri A., Shao W., Wiegand A., Simonetti F.R., Mellors J.W., Hughes S.H., Coffin J.M., Maldarelli F, & Kearney M.F. (2019). HIV Infected T Cells Can Proliferate in vivo Without Inducing Expression of the Integrated Provirus. Frontiers in Microbiology, 10, 2204.

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Quantification of Tetramer-Specific CD8+ T Cells

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PBMC and tissue-derived MNC were thawed and subjected to Live/Dead Aqua fluorescent reactive dye (ThermoFisher) in PBS for 30 mint at RT. The cells were washed cells with RPMI + 10% FBS and subsequently incubated with 3µg/mL A01-CM9 tetramers (NIH Tetramer Core Facility and MBL International) in PBS + 2% FBS at room temperature for 35 min at 2 × 10⁶ cells/100 µL in polypropylene FACS tubes. The cells were then incubated at 4 ^°C with antibodies directed to cell surface markers for additional 30 min. After 2 washes with PBS + 2% FBS, the cells were fixed with 200 µL of stabilizing fixative (BD Biosciences) at room temperature. Tetramer + CD8 + T cells were quantified with the aid of a Fortessa flow cytometer (BD) by gating on live, singlets, CD45 + , CD8 + CD3 + CD14- CD20- CD4- cells present in the lymphocyte gate (CD45-PerCP, BD Biosciences, catalog number 558411, 1:10 dilution; CD8-BUV395, BD Biosciences, catalog number 563795, 1:50 dilution; CD3-FITC BD Biosciences, catalog number 556611, 1:5 dilution; CD14-PE-Cy7, BD Biosciences, catalog number 557742, 1:100 dilution; CD20-PE-Cy7, BD Biosciences, catalog number 560735, 1:50 dilution; CD4-BV785, Biolegend, catalog number 317442, 1:100 dilution).

Colonna L., Peterson C.W., Schell J.B., Carlson J.M., Tkachev V., Brown M., Yu A., Reddy S., Obenza W.M., Nelson V., Polacino P.S., Mack H., Hu S.L., Zeleski K., Hoffman M., Olvera J., Furlan S.N., Zheng H., Taraseviciute A., Hunt D.J., Betz K., Lane J.F., Vogel K., Hotchkiss C.E., Moats C., Baldessari A., Murnane R.D., English C., Astley C.A., Wangari S., Agricola B., Ahrens J., Iwayama N., May A., Stensland L., Huang M.L., Jerome K.R., Kiem H.P, & Kean L.S. (2018). Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation. Nature Communications, 9, 4438.

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Isolation and Characterization of CD4+ T Cells

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Participant recruitment and informed consent were performed under Institutional Review Board (IRB)-approved protocols at the US National Institutes of Health (NIH) and University of Washington. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation. PBMC were incubated with Fcγ-receptor-blocking reagent for 10 minutes and stained with LIVE/DEAD Aqua stain, CD3-APC-H7 (BD Biosciences; Cat# 641406), CD4-BV785 (BioLegend; Cat# 317442), CD8-PacBlue (Invitrogen; Cat# MHCD0828), CD14-BV650 (BioLegend; Cat# 301836), CD16-PerCP/Cy5.5 (BioLegend; Cat# 302028), CD19-BV605 (BD Biosciences; Cat# 562653), CD20-BV570 (BioLegend; Cat# 302332), CD27-Alx700 (BioLegend; Cat# 302814), CD32-PE (BioLegend; Cat# 303206); CD45RO-ECD (Beckman Coulter; Cat# IM2712U), CD123-PE/Cy5 (BD Biosciences; Cat# 551065), and TCRγδ-APC (BD Biosciences; Cat# 555718). CD4+ T cells were isolated by fluorescence-activated cell sorting (FACS) on a FACSAria (Becton Dickinson) using previously described protocols^{32 (link)}.

Sun C., Liu L., Pérez L., Li X., Liu Y., Xu P., Boritz E.A., Mullins J.I, & Abate A.R. (2022). Droplet microfluidic sequencing of HIV proviruses with their integration sites in patients receiving antiretroviral therapy. Nature biomedical engineering, 6(8), 1004-1012.

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Comprehensive Murine and Human Immune Profiling

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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).

Sharon S., Duhen T., Bambina S., Baird J., Leidner R., Bell B., Casap N., Crittenden M., Vasudevan S., Jubran M., Kravchenko-Balasha N, & Gough M. (2021). Explant Modeling of the Immune Environment of Head and Neck Cancer. Frontiers in Oncology, 11, 611365.

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Comprehensive Lung and Spleen Cell Analysis

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Lung tissue was chopped and digested with collagenase-D (1 mg/ml; Roche) and DNase I (10 mg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. To detect cytokines, brefeldin A (5 µg/ml) was added during the digest. Lungs or spleens were passed through a 70-µm cell strainer to a obtain single-cell suspension, followed by RBC lysis with ACK buffer. The cells were incubated with Fcγblock (anti-CD16/CD32 antibody, BD Biosciences) (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. The following surface Abs were used: CD69-FITC, CD3-APC-ef780, MHCII-APC, Ly6C-PerCP-Cy5.5, CD86-FITC, CD11b-APC-ef780, CD38-ef450, F4/80-PE-Cy5, CD49d-PerCP-ef710, CD3-AF700, CD8-APC-ef780, CD44-PE-Cy7, CD4-PE-Cy5 (eBiosciences), CD44-BV605, CD4-BV785, CD103-PE, Ly6G-BV605, CD80-PE-Dazzle594 (BioLegend), CD62L-PE-CF594, SiglecF-PE, CD103-BV786 (BD Biosciences). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 (BD Biosciences). Fluorescence minus one samples were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

Borkner L., Misiak A., Wilk M.M, & Mills K.H. (2018). Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory. Frontiers in Immunology, 9, 1764.

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