Cd4 clone okt4

Manufactured by BioLegend

CD4 (clone OKT4) is a monoclonal antibody that recognizes the CD4 antigen, a membrane glycoprotein expressed on the surface of T helper cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry applications.

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Lab products found in correlation

Cd3 clone sp34 2, by BD (8 mentions) Cd20 clone 2h7, by BioLegend (5 mentions) Cd8 clone rpa t8, by BioLegend (3 mentions) Igg clone g18 145, by BD (3 mentions) Cd8 clone rpa t8, by BD (3 mentions) Cd8 clone hit8a, by BioLegend (2 mentions) Igm clone g20 127, by BD (2 mentions) Igg g18 145, by BD (2 mentions) Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (2 mentions) Cd14 clone m5e2, by BioLegend (2 mentions) Cd14 clone m5e2, by BD (2 mentions) Anti rhesus igg h l, by Bio-Rad (2 mentions) Mycozap plus pr, by Lonza (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Ifn γ clone b27, by BD (2 mentions) Cd8 clone sk1, by BD (2 mentions) Pe conjugated anti cd3 clone sp34, by BD (1 mentions) Hla dr clone l243, by BioLegend (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Dynabeads human t activator cd3 cd28 for t cell expansion and activation, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

CD4 (OKT4, (19 citations) Anti-CD4 (clone OKT4, (12 citations) CD4-PerCp-Cy5.5 clone OKT4 (3 citations) Anti-CD4-FITC(Clone OKT4, (3 citations) CD4-Brilliant Violet 650 (clone OKT4; (3 citations) CD4-PE (clone OKT4, (2 citations) CD4-BV421 (clone OKT4, (2 citations) CD4-BV785 (clone OKT4) (2 citations) CD4-PerCP (clone OKT4, (2 citations) Anti-human CD4-allophycocyanin (clone: OKT4) (2 citations)

11 protocols using cd4 clone okt4

Flow Cytometry Analysis of T Cells and Monocytes

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All CD11b⁺ myeloid cells were labeled with PE-conjugated anti-CD3 clone SP34 (BD Bioscience) and fluorescein isothiocyanate-conjugated anti-CD11b clone Bear1 (Beckman Coulter, Brea, CA) to assess the selection efficiency. Purified CD4⁺ T cells were stained with antibodies for HLA-DR clone L243 (BioLegend), CD3 clone SP34-2 (BD Bioscience), CD4 clone OKT4 (BioLegend), CD8 clone RPA-T8 (BioLegend), and TCRγδ clone B1.1 (eBioscience). Cells were stained for 20 min at room temperature in 100 μl phosphate-buffered saline–2% FBS and fixed for 10 min with Fix/Lyse buffer (Becton Dickinson, Franklin Lakes, NJ). After fixation, samples were analyzed in a BD LSRFortessa flow cytometer using DIVA software (Becton Dickinson, Franklin Lakes, NJ). The gating of CD3⁺ T cells was easily visualized as small CD3⁺ nonautofluorescent cells. All data were analyzed using FlowJo software. CD4⁺ T cell and monocyte counts were analyzed as previously described (38 (link)).

Avalos C.R., Price S.L., Forsyth E.R., Pin J.N., Shirk E.N., Bullock B.T., Queen S.E., Li M., Gellerup D., O'Connor S.L., Zink M.C., Mankowski J.L., Gama L, & Clements J.E. (2016). Quantitation of Productively Infected Monocytes and Macrophages of Simian Immunodeficiency Virus-Infected Macaques. Journal of Virology, 90(12), 5643-5656.

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Expansion and Activation of T Cells

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For T cell activation, PBMCs were cultured for 5 days with anti-human CD3/CD28-coated beads (Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation; #11132D; Invitrogen) and 5 ng/mL human recombinant IL-12 (#573002; BioLegend). Human recombinant IL-2 (#589106; BioLegend) at 30 U/mL and IL-15 (#570304; BioLegend) at 5 ng/mL was added on day 2 and the culture continued. On day 5, beads were removed and the cells were maintained in culture for another 48 h with IL-2 (10 U/mL) and IL-15 (5 ng/mL). Cells were stained with fluorochrome-labeled antibodies to CD4 (clone OKT4; #317432; BioLegend), CD8 (clone HIT8a; #300918; BioLegend) and LAG-3 (clone 11C3C65; #369326; BioLegend) and analyzed by flow cytometry or used in functional assays.

Baleeiro R.B., Bouwens C.J., Liu P., Di Gioia C., Dunmall L.S., Nagano A., Gangeswaran R., Chelala C., Kocher H.M., Lemoine N.R, & Wang Y. (2022). MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy. Oncoimmunology, 11(1), 2080329.

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Isolation and Expression of Antigen-Specific Rhesus Macaque Antibodies

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Cryopreserved rhesus macaque PBMCs were thawed and stained with LIVE/DEAD fixable violet dead cell stain (Life Technologies). After washing, cells were stained with a cocktail of anti-human antibodies, including CD3 (clone SP34–2; BD Biosciences), CD4 (clone OKT4; BioLegend), CD8 (clone RPA-T8; BioLegend), CD14 (clone M5E2; BioLegend), CD20 (clone 2H7; BioLegend), IgG (G18–145; BD Biosciences), and IgM (clone G20–127; BD Biosciences), and with fluorescently labeled trimer probe (BG505 DS-SOSIP.avi) and peptide probe (FP10–1M6T.avi or FP9-PEG12-biotin) (Kong et al., 2016 (link)). Vivid-CD3-CD4-CD8-CD14-CD20+IgG+IgM- memory B cells that are positively stained with both trimer and peptide probes were sorted into 96-well plates containing lysis solution as previously described (Kong et al., 2016 (link)). Nested PCR was performed using published primers (Mason et al., 2016 (link); Sundling et al., 2012 (link)). Heavy and light chain sequences were cloned into expression vectors containing rhesus macaque immunoglobulin constant regions. IgG was expressed by cotransfecting Expi293F™ cells with equal amounts of paired heavy and light chain plasmids and purified using protein A Fast Flow (GE Healthcare) according to the manufacturer’s instructions.

Kong R., Duan H., Sheng Z., Xu K., Acharya P., Chen X., Cheng C., Dingens A.S., Gorman J., Sastry M., Shen C.H., Zhang B., Zhou T., Chuang G.Y., Chao C.W., Gu Y., Jafari A.J., Louder M.K., O’Dell S., Rowshan A.P., Viox E.G., Wang Y., Choi C.W., Corcoran M.M., Corrigan A.R., Dandey V.P., Eng E.T., Geng H., Foulds K.E., Guo Y., Kwon Y.D., Lin B., Liu K., Mason R.D., Nason M.C., Ohr T.Y., Ou L., Rawi R., Sarfo E.K., Schön A., Todd J.P., Wang S., Wei H., Wu W., Mullikin J.C., Bailer R.T., Doria-Rose N.A., Hedestam G.B., Scorpio D.G., Overbaugh J., Bloom J.D., Carragher B., Potter C.S., Shapiro L., Kwong P.D, & Mascola J.R. (2019). Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization. Cell, 178(3), 567-584.e19.

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Isolation and Expansion of SIV-Reactive Memory B Cells

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Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail (1:100 dilution) of CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled biotinylated SIVmac239 SOSIP.664 Env at room temperature for 20 min in the dark. SIVmac239 Env trimer probes were labeled with two different fluorophores, from which SIVmac239 Env dual positive memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺SIVmac239 SOSIP²⁺) were analyzed with BD FACSMelody or BD FACSFusion, and single-cell sorted, cultured, expanded in 384-well plates as described previously^{28 (link),29 (link)}. Briefly, sorted B cells were cultured with Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× MycoZap Plus-PR (Lonza), 100 U/mL human IL-2 (Roche), 50 ng/mL human IL-21 (Invitrogen), 50 ng/mL human IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H + L) (BioRad), and irradiated 3T3msCD40L feeder cells. Flow cytometric data were subsequently analyzed using FlowJo (v10.7.1).

Zhao F., Berndsen Z.T., Pedreño-Lopez N., Burns A., Allen J.D., Barman S., Lee W.H., Chakraborty S., Gnanakaran S., Sewall L.M., Ozorowski G., Limbo O., Song G., Yong P., Callaghan S., Coppola J., Weisgrau K.L., Lifson J.D., Nedellec R., Voigt T.B., Laurino F., Louw J., Rosen B.C., Ricciardi M., Crispin M., Desrosiers R.C., Rakasz E.G., Watkins D.I., Andrabi R., Ward A.B., Burton D.R, & Sok D. (2022). Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus. Nature Communications, 13, 5236.

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Flow Cytometric Analysis of T Cell Activation

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Flow cytometric analysis was conducted as previously described [12 (link)]. Intracellular cytokine staining was performed to evaluate T cell activation. Conjugated mouse monoclonal antibodies specific for the following determinants were used: CD4 (clone OKT4; BioLegend, San Diego, CA), CD8a (clone RPA-T8; BioLegend), CD137 (clone 4B4-1; BD Biosciences, San Diego, CA), CD154 (clone TRAP1; BD Biosciences), and IFN-γ (clone B27; BD Biosciences). Appropriate isotype controls were included in each analysis.

Tomimaru Y., Mishra S., Safran H., Charpentier K.P., Martin W., De Groot A.S., Gregory S.H, & Wands J.R. (2015). Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma. Vaccine, 33(10), 1256-1266.

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Multiparameter Phenotyping of Immune Cells

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Cells were stained with fluorochrome-conjugated Abs specific for CXCR5 (clone MU5UBEE; eBioscience), CD3 (clone SP-34-2; BD Biosciences), CXCR3 (clone IC6; BD Biosciences), CCR6 (clone 11A9), CCR4 (clone 1G1), ICOS (clone C398.4A; ebioscience), CD95 (clone; BD Biosciences), PD-1(Clone EH12.1; Biolegend), CD8 (Clone SK1; BD Bioscience), CD4 (clone; OKT4; biolegend), CCR5 (Clone 3A9, BD Biosciences), α4β7 (Act1, NHP reagent resource), Live Dead-IR stain (Invitrogen), Ki-67 (Clone B56; BD Biosciences), Bcl-6 (clone K112-91), IL-21 (Clone 3A3-N2; BD Biosciences), CD4 (Clone L200; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences). FITC conjugated CD40L (Clone TRAP1; BD Biosciences).

Velu V., Mylvaganam G.H., Gangadhara S., Hong J.J., Iyer S.S., Gumber S., Ibegbu C.C., Villinger F, & Amara R.R. (2016). Induction of Th1 biased Tfh (Tfh1 cells) in lymphoid tissues during chronic SIV infection defines functionally distinct germinal center Tfh cells. Journal of immunology (Baltimore, Md. : 1950), 197(5), 1832-1842.

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Isolation and Expansion of Env-specific Memory B Cells

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Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail of CD3 (clone SP34-2, BD Biosciences), CD4(clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (clone G20-127, BD Biosciences or clone MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled BG505 Env at room temperature for 20 min in the dark. BG505 SOSIP.664 was labeled with two fluorophores separately, from which dual positive Env-specific (CD3^-CD4^-CD8^-CD14^-CD20⁺IgM^-IgG⁺BG505 SOSIP²⁺) and/or 398F1 SOSIP⁺ memory B cells were analyzed with a FACSFusion sorter and then single-cell sorted, cultured, and expanded as previous described (Huang et al., 2013 (link)). In brief, cells were sorted into Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1X MycoZap Plus-PR (Lonza), 100 U/mL IL-2 (Roche), 50 ng/mL IL-21 (Invitrogen), 50 ng/mL IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H+L) (BioRad) and irradiated 3T3msCD40L feeder cells.

Zhao F., Joyce C., Burns A., Nogal B., Cottrell C.A., Ramos A., Biddle T., Pauthner M., Nedellec R., Qureshi H., Mason R., Landais E., Briney B., Ward A.B., Burton D.R, & Sok D. (2020). Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques. Cell Reports, 32(10), 108122.

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Identifying Dual-Specific Memory B Cells

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FACS analysis of PBMC was performed as described previously (Kong et al., 2019 (link)). Briefly, NHP PBMCs were stained with LIVE/DEAD fixable violet dead cell stain (Life Technologies), washed, and then stained with a cocktail of anti-human antibodies, including CD3 (clone SP34–2; BD Biosciences), CD4 (clone OKT4; BioLegend), CD8 (clone RPA-T8; BioLegend), CD14 (clone M5E2; BioLegend), CD20 (clone 2H7; BioLegend), IgG (G18–145; BD Biosciences), and IgD (Dako, polyclonal), and with fluorescently labeled trimer probe (BG505 DS-SOSIP) for 15 mins, and followed by peptide probe FP9-PEG12-PE for another 15 mins. Stained PBMCs were analyzed on BD LSRFortessa X-50. Vivid−CD3−CD4−CD8−CD14−CD20+IgG+IgD− memory B cells that were positively stained with both trimer and peptide probes were considered FP and trimer dual-specific memory B cells. The analysis of the PBMCs was performed using FlowJo.

Cheng C., Duan H., Xu K., Chuang G.Y., Corrigan A.R., Geng H., O’Dell S., Ou L., Chambers M., Changela A., Chen X., Foulds K.E., Sarfo E.K., Jafari A.J., Hill K.R., Kong R., Liu K., Todd J.P., Tsybovsky Y., Verardi R., Wang S., Wang Y., Wu W., Zhou T., Arnold F.J., Doria-Rose N.A., Koup R.A., McDermott A.B., Scorpio D.G., Worobey M., Shapiro L., Mascola J.R, & Kwong P.D. (2020). Immune Monitoring Reveals Fusion Peptide Priming to Imprint Cross-Clade HIV-Neutralizing Responses with a Characteristic Early B Cell Signature. Cell reports, 32(5), 107981.

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Multiparametric Immune Phenotyping

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Cells were stained with fluorochrome-conjugated Abs specific for CD3 (clone SP-34-2; BD Biosciences), CCR6 (clone 11A9), CD95 (clone DX2; BD Biosciences), PD-1 (Clone EH12.1; Biolegend), CD8 (Clone SK1; BD Bioscience), CD4 (clone; OKT4; Biolegend), α4β7 (Act1, NHP reagent resource), LIVE/DEAD fixable Near-IR Dead Cell stain (Invitrogen), Ki-67 (Clone B56; BD Biosciences), IFN-γ (Clone B27; BD Biosciences), CD95 (clone DX2; BD Biosciences), CD28 (Clone 28.2; BD Biosciences), IL-17A (eBio64DEC17; eBioscience), TNF-α (MAb11; BD biosciences), IL-22 (IL22JOP; ebiosciences), IL-7R (Clone eBioRDR5; eBioscience), CD161 (HP3G10; Biolegend), TCR7.2 (3C10; Biolegend), CD69 (FN50; Biolegend), IL-18Rα (H44; Biolegend), streptavidin-BV421- conjugated MR1 tetramer (NIH tetramer core facility), Granzyme B (Clone GB1; BD Biosciences 1), and perforin (Clones Pf-80/164; BD Biosciences).

Murugesan A., Ibegbu C., Styles T.M., Jones A.T., Shanmugasundaram U., Reddy P.B., Rahman S.J., Saha P., Vijay-Kumar M., Shankar E.M., Amara R.R, & Velu V. (2020). Functional MAIT Cells Are Associated With Reduced Simian–Human Immunodeficiency Virus Infection. Frontiers in Immunology, 10, 3053.

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Isolation and Screening of Antigen-Specific Memory B Cells

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Single cell sorting was performed as previously described (Mason et al., 2016 (link)). In brief, rhesus PBMCs were stained with an antibody cocktail of CD3 (clone SP34–2, BD Biosciences), CD4 (clone OKT4, BioLegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (clone MHM-88, Biolegend), IgG (clone G18–145, BD Biosciences) and BG505 SOSIP-AviB conjugated with streptavidin-Alexa Fluor 647 (AF647) and streptavidin-Alexa Fluor 488 (AF488) respectively. Cells were stained at room temperature for 20 mins in the dark. Antigen-specific memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺BG505 SOSIP.664 dual positive) were single-cell sorted into 384-well culture plates according to the published protocol (Huang et al., 2013 (link)). After 2 weeks of culture, supernatants were harvested and screened for neutralization activity. Ig genes from neutralization positive wells were amplified by RT-PCR, nested PCR, then cloned into expression vectors and expressed in 293F cells.

Nogal B., Bianchi M., Cottrell C.A., Kirchdoerfer R.N., Sewall L.M., Turner H.L., Zhao F., Sok D., Burton D.R., Hangartner L, & Ward A.B. (2020). Mapping Polyclonal Antibody Responses in Non-human Primates Vaccinated with HIV Env Trimer Subunit Vaccines. Cell reports, 30(11), 3755-3765.e7.

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