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Anti cd4 clone okt4

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Anti-CD4 (clone OKT4) is a monoclonal antibody that binds to the CD4 receptor expressed on the surface of T helper cells. The antibody is a useful tool for the identification and study of CD4+ T cells in various applications, such as flow cytometry and cell-based assays.

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Lab products found in correlation

Lsr 2, by BD (4 mentions) Anti pd 1 clone 29f 1a12, by BioLegend (3 mentions) Anti tigit clone 1g9, by BioLegend (3 mentions) Anti cd4 clone rm4 5, by BioLegend (3 mentions) Anti tigit clone a15153g, by BioLegend (3 mentions) Human cd25 clone bc96, by BioLegend (3 mentions) Human foxp3 clone 236a e7, by BioLegend (3 mentions) Human pd 1 clone eh12.2h7, by BioLegend (3 mentions) Facsaria 2, by BD (2 mentions) Ccr6 clone go34e3, by BioLegend (2 mentions) Anti ccr7 clone go43h7, by BioLegend (2 mentions) Spectroflo software, by Cytek (2 mentions) Anti cd16 clone 3g8, by BioLegend (2 mentions) Anti cd14 clone m5e2, by BioLegend (2 mentions) Anti cd25 clone pc61, by BioLegend (1 mentions) Anti pd l1 clone 10f 9g2, by BioLegend (1 mentions) Anti 2b4 clone m2b4 b6 458, by BioLegend (1 mentions) Anti tim3 clone rmt3 23, by BioLegend (1 mentions) Anti cd25 clone bc96, by BioLegend (1 mentions) Anti pd 1 clone eh12.2h7, by BioLegend (1 mentions)

12 protocols using anti cd4 clone okt4

1

Sorting Memory CD4+ T Cells

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Mononuclear cells isolated from the blood and pooled lymph nodes (axillary, inguinal and mesenteric) of chronically SIV-infected RMs were stained with anti-CD3, anti-CD4 (clone OKT4; BV650, Biolegend), anti-CD28, anti-CD95, anti-CCR6, and anti-CD161. Memory CD4+ T cells were then sorted based on their expression, or lack of expression, of CCR6 and CD161 using a FACS AriaII (BD Biosciences).
McGary C.S., Alvarez X., Harrington S., Cervasi B., Ryan E.S., Iriele R.I., Paganini S., Harper J., Easley K., Silvestri G., Ansari A.A., Lichterfeld M., Micci L, & Paiardini M. (2017). The loss of CCR6+ and CD161+ CD4+ T cell homeostasis contributes to disease progression in SIV-infected rhesus macaques. Mucosal immunology, 10(4), 1082-1096.
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2

Evaluating Immune Checkpoint Expression

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Mouse spleen cells were washed with PBS containing 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-GITR (clone yGITR765, Biolegend), anti-CD25 (clone PC61, Biolegend), anti-PD-L1 (clone 10F.9G2, Biolegend), anti-CTLA4 (clone UC10-4B9, Biolegend), anti-2B4 (clone M2B4(B6)458.1, Biolegend), anti-VISTA (clone MIH63, Biolegend), anti-LAG3 (clone C9B7W, Biolegend), anti-TIM3 (clone RMT3-23, Biolegend), and anti-CD4 (clone RM4-5, Biolegend).
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), anti-CD25 (clone BC96, Biolegend), anti-FoxP3 (clone 236A/E7, Biolegend), anti-PD-1 (clone EH12.2H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.
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3

Flow Cytometry Analysis of Mouse and Human Immune Cells

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Staining of mouse cells for flow cytometry analysis was done after cells were collected and washed with PBS with 1% BSA (staining buffer), and then Fc-blocked with mouse IgG. Conjugated antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-CCR6 (clone 29-2L17), anti-CCR7 (clone 4B12), anti-CXCR6 (clone SA051D1). Mouse spleen cells were washed with staining buffer (PBS + 1% BSA), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies.
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236A/E7, Biolegend), human PD-1 (clone EH12.2H7, Biolegend), CCR6 (clone GO34E3, Biolegend), anti-CCR7 (clone GO43H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences, Franklin Lakes, NJ) or in the Ocular Immunobiology P30 Core Module at Dean McGee Eye Institute on a Cytek Aurora (Cytek, Fremont, CA). Spectral unmixing of full spectrum flow cytometry data was done with SpectroFlo software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).
Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.
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4

TCR Engineering for Adoptive Therapy

Cited 1 time
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Full-length TCR α and β chain genes were cloned by 5’-RACE PCR method and sequenced as previously described [28 (link)]. TCR β chain gene was genetically fused to α chain gene via T2A site and cloned into a murine stem cell virus-derived retroviral vector. Retroviral vector production and transduction were performed as previously described [26 (link)]. Transduction efficiency was determined by staining with A*02/NY-ESO-1157-165 (SLLMWITQC) tetramer (iTAg MHC tetramer, MBL), anti-human TCR Vβ8 mAb (clone JR2, BioLegend) for 19305DP-TCR, and anti-human TCR Vβ3 mAb (clone CH92, Beckman Coulter) for CD8SP (CD8SP1) or control HLA-DR*01:01 (DR*01)-restricted NY-ESO-1-specific TCR [29 (link)]. In some experiments, CD4+ or CD8+ T cells were depleted from PBMC by anti-biotin magnetic beads (Dynabeads, Life Technologies) following staining with biotin-conjugated anti-CD4 (clone OKT4, BioLegend) or anti-CD8 (clone HIT8a, BioLegend) mAb prior to activation with PHA. For in vivo experiments, T cells were harvested 20–22 h after the second infection.
Matsuzaki J., Tsuji T., Chodon T., Ryan C., Koya R.C, & Odunsi K. (2019). A rare population of tumor antigen-specific CD4+CD8+ double-positive αβ T lymphocytes uniquely provide CD8-independent TCR genes for engineering therapeutic T cells. Journal for Immunotherapy of Cancer, 7, 7.
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5

Flow Cytometry Analysis of Cytokine Production

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PBMCs were suspended in FACS buffer (PBS, 2% FCS, 1% sodium azide) at 1×106 per well of 96 well culture plates and incubated for 4h in the presence of brefeldin A (Sigma) with or without PMA (70ng/ml) and ionomycin (700ng/ml). For evaluation of surface receptors, cells were stained with the viability Ghost Dye violet 510 (Tonbo) and monoclonal antibodies including anti-CD3 clone SKY7 (Biolegend) and anti-CD4 clone OKT4 (Biolegend). Cells were incubated with antibodies at room temperature for 20 min and then washed with FACS buffer. For intracellular staining cells were permeabilized with Cytofix/Cytoperm reagent (BD) and incubated for 30 min at room temperature after incubation with anti-IFN-γ clone 4S.B3 (eBioscience), anti-IL17A clone eBio64CAP17 (eBioscience) or anti-IL-13 clone PVM13-1 (eBioscience). Samples were acquired using a Fortessa flow cytometer (BD Bioscience) and analyzed using FlowJo software
Watkin L.B., Jessen B., Wiszniewski W., Vece T., Jan M., Sha Y., Thamsen M., Santos-Cortez R.L., Lee K., Gambin T., Forbes L., Law C.S., Stray-Petersen A., Cheng M.H., Mace E.M., Anderson M.S., Liu D., Tang L.F., Nicholas S.K., Nahmod K., Makedonas G., Canter D., Kwok P.Y., Hicks J., Jones K.D., Penney S., Jhangiani S.N., Rosenblum M.D., Dell S.D., Waterfield M.R., Papa F.R., Muzny D.M., Zaitlen N., Leal S.M., Gonzaga-Jauregui C., Boerwinkle E., Eissa N.T., Gibbs R.A., Lupski J.R., Orange J.S, & Shum A.K. (2015). COPA mutations impair ER-Golgi transport causing hereditary autoimmune-mediated lung disease and arthritis. Nature genetics, 47(6), 654-660.
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6

Monitoring Donor Chimerism via Multimodal Analysis

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Donor chimerism was monitored by molecular chimerism analysis-based divergent microsatellite markers as previously described30 (link),31 (link), as well as by flow cytometry using a discriminatory anti-Mamu A01 antibody34 (link). Flow cytometric analysis was performed with FlowJo (Tree Star) using the following analysis pipeline: (1) Doublets were excluded on the basis of FSC-H vs. FSC-A and SSC-H vs. SSC-A gating, (2) Neutrophils cells were identified as high SSC, CD11b + CD3- cells, (3) T cells as CD3 + CD20- CD11b- cells present in the lymphocyte gate (then sub-gated for CD4 or CD8 positivity), and (4) B cells as CD3- CD20 + lymphocytes. The following antibody clones were used for this analysis: anti-Mamu A01 clone P1234 (link) (Anti-Mamu A01-PE, NHP Reagent Resources,1:50 dilution), anti-CD11b clones ICRF44 and D12 (BD Biosciences, catalog numbers 557918 and 340936, 1:100 dilution), anti-CD3 clone SP34–2 (BD Biosciences, catalog number 557757, 1:50 dilution), anti-CD4 clone OK-T4 (Biolegend, catalog number 317442, 1:100), anti-CD8 clone RPA-T8 (BD Biosciences, catalog number 563795, 1:50 dilution), anti-CD20 clone 2H7 (ThermoFisher, catalog number 45-0209-42, 1:50 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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7

Multiparameter Flow Cytometry Profiling of PBMCs

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Human PBMCs were stained with anti-CD14 (clone M5E2, Biolegend), anti-CD16 (clone 3G8, Biolegend), anti-CD4 (clone OKT4, Biolegend), human CD39 (clone 498403, R&D Systems), human CD73 (clone 606112, R&D Systems)44 (link),45 (link), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236 A/E7, Biolegend), and human PD-1 (clone EH12.2H7, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR). Single stained samples were used to determine compensation values, and fluorescence minus one (FMO) controls were used to determine gating placement.
Muhammad F., Wang D., Montieth A., Lee S., Preble J., Foster C.S., Larson T.A., Ding K., Dvorak J.D, & Lee D.J. (2019). PD-1+ melanocortin receptor dependent-Treg cells prevent autoimmune disease. Scientific Reports, 9, 16941.
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8

Multiparameter Analysis of CAR T Cells

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CAR T cells were stained with a Live/Dead Fixable Near-IR stain (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, followed by a phosphate-buffered saline (PBS) wash and incubation with a ChromPure Human IgG blocking solution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 5 min. An antibody cocktail containing anti-PSMA antibody (clone LNI-17; BioLegend, San Diego, CA, USA), anti-EGFRt (Erbitux; Lilly, Indianapolis, IN, USA), anti-CD4 (clone OKT4; BioLegend), anti-CD8 (clone RPA-T8; BioLegend), and an in-house–generated anti-idiotypic antibody to detect the CAR was added for 20 min. Cells were then washed twice with staining buffer (BioLegend) and analyzed on a NovoCyte cytometer (ACEA Biosciences, San Diego, CA, USA). Data analysis was performed using FlowJo v10.1 software (Tree Star, Ashland, OR, USA).
Minn I., Huss D.J., Ahn H.H., Chinn T.M., Park A., Jones J., Brummet M., Rowe S.P., Sysa-Shah P., Du Y., Levitsky H.I, & Pomper M.G. (2019). Imaging CAR T cell therapy with PSMA-targeted positron emission tomography. Science Advances, 5(7), eaaw5096.
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9

Flow Cytometric Analysis of Treg Cells

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Foxp3/Transcription Factor Staining Buffer Set, True‐Phos™ Perm Buffer, Cell Staining Buffer, and recombinant human cytokines for in vitro stimulation (IFN‐gamma, interleukin (IL)‐6, IL‐2, IL‐4; all carrier‐free) were purchased from Biolegend.
The following antibodies were used to analyse Tregs via flow cytometry: anti‐CD4 (clone OKT4; APC‐Cy7‐labeled), anti‐CD3 (clone HIT3a; PE‐Cy7) anti‐CD25 (clone M‐A251; PerCP‐labeled), anti‐CD127 (clone A019D5; BV‐421‐labeled), anti‐Foxp3 (clone 206D; AlexaFluor647‐labeled), anti‐CD39 (clone A1; FITC‐labeled), and isotype controls (all from Biolegend). STAT activation was studied using commercially available PE‐labeled antibodies against phospho‐STAT1 (clone A17012A.Rec), phospho‐STAT3 (clone 13A3‐1), phospho‐STAT5 (clone A17016B.Rec), and phospho‐STAT6 (clone A15137E) (all Biolegend).
Dold L., Kalthoff S., Frank L., Zhou T., Esser P., Lutz P., Strassburg C.P., Spengler U, & Langhans B. (2024). STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis. Immunity, Inflammation and Disease, 12(4), e1248.
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10

Flow Cytometry Analysis of Mouse and Human Immune Cells

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Staining of mouse cells for flow cytometry analysis was done after cells were collected and washed with PBS with 1% BSA (staining buffer), and then Fc-blocked with mouse IgG. Conjugated antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-CD4 (clone RM4-5, Biolegend), anti-CCR6 (clone 29-2L17), anti-CCR7 (clone 4B12), anti-CXCR6 (clone SA051D1). Mouse spleen cells were washed with staining buffer (PBS + 1% BSA), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies.
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236A/E7, Biolegend), human PD-1 (clone EH12.2H7, Biolegend), CCR6 (clone GO34E3, Biolegend), anti-CCR7 (clone GO43H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences, Franklin Lakes, NJ) or in the Ocular Immunobiology P30 Core Module at Dean McGee Eye Institute on a Cytek Aurora (Cytek, Fremont, CA). Spectral unmixing of full spectrum flow cytometry data was done with SpectroFlo software (Cytek) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).
Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.
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