Clone okt 3

Manufactured by BioXCell

Clone OKT-3 is a monoclonal antibody that binds to the CD3 epsilon chain of the T cell receptor complex. It is used as a research tool for the activation and expansion of T cells in vitro.

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Lab products found in correlation

Clone cd28, by BioLegend (3 mentions) Anti cd3, by BioXCell (3 mentions) Clone pv 1, by BioXCell (3 mentions) Anti cd28, by BioXCell (3 mentions) Positive selection, by Miltenyi Biotec (2 mentions) Elisa, by R&D Systems (2 mentions) Elisa, by Thermo Fisher Scientific (1 mentions) Optimem glutamax, by Thermo Fisher Scientific (1 mentions) Dmxaa, by InvivoGen (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Clone mopc 21, by BioXCell (1 mentions) Cd16 apc cy7, by BD (1 mentions) Clone fn50, by BD (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Clone cd28, by BD (1 mentions) Efluor 670, by BD (1 mentions) Clone rpa t4, by BD (1 mentions) Clone sk1, by BD (1 mentions) Clone sk7, by Thermo Fisher Scientific (1 mentions) Facs lsr 2, by BD (1 mentions)

7 protocols using clone okt 3

Cytokine Production by Activated PBMCs

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PBMCs were obtained from human buffy coats by density gradient centrifugation using Ficoll-Paque. The cells (5 x 10⁵ PBMCs/well) were stimulated with antibodies against CD3 (1 μg/ml, clone OKT-3, BioXcell) and CD28 (1 μg/ml, clone CD28.2, BioLegend) in the presence of human IL-39 IgG1-Fc fusion or IgG1-Fc control protein (3 μg/ml). Supernatants were collected and IFNγ or IL-17A cytokines were quantified by ELISA (eBioscience) after 48 hrs or 72 hrs of incubation, respectively.

Ecoeur F., Weiss J., Schleeger S, & Guntermann C. (2020). Lack of evidence for expression and function of IL-39 in human immune cells. PLoS ONE, 15(12), e0242329.

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Transfection of Cyclic Dinucleotides in CD4 T Cells

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Mouse naive CD4 T cells were seeded on plate-bound anti-CD3 (2 µg/mL, clone 17A2; BioXCell) and anti-CD28 (2 µg/mL, clone PV1; BioXCell). Human naive CD4 T cells were seeded on plate-bound anti-CD3 (5 μg/mL, clone OKT-3; BioXCell) and anti-CD28 (2 µg/mL, clone CD28.2; BioLegend). Cells (3×10⁶ cells/mL) were transfected for 6 hours with 80 µg/L cyclic dinucleotides (unless specified otherwise) using Opti-MEM Glutamax (Gibco) and Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Alternatively, 1.5×10⁶ cells/mL CD4 T cells were treated with indicated doses of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) for 4 hours in mouse T-cell culture medium.
2′3′-cGAMP (cyclic (G(2′,5′)pA(3′,5′)p)), referred to as cGAMP; 2′3′-cGAMP control (2′5′-GpAp), referred to as control; as well as DMXAA (Murine STING ligand, Xanthenone Analog) were purchased from InvivoGen.
When indicated, mTOR inhibition was achieved by adding 10 nM of rapamycin (Rapa; Calbiochem, Merck) during the 6-hour transfection step.

Benoit-Lizon I., Jacquin E., Rivera Vargas T., Richard C., Roussey A., Dal Zuffo L., Martin T., Melis A., Vinokurova D., Shahoei S.H., Baeza Garcia A., Pignol C., Giorgiutti S., Carapito R., Boidot R., Végran F., Flavell R.A., Ryffel B., Nelson E.R., Soulas-Sprauel P., Lawrence T, & Apetoh L. (2022). CD4 T cell-intrinsic STING signaling controls the differentiation and effector functions of TH1 and TH9 cells. Journal for Immunotherapy of Cancer, 10(1), e003459.

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CD11b+ Cell-Mediated T Cell Suppression

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Tumors were digested as described above, and CD11b⁺ cells were isolated magnetically by positive selection (Miltenyi). Purified CD11b⁺ cells were combined at indicated ratios with RBC-depleted C57BL/6 mouse splenocytes, and added to a 96-well plate pre-coated with anti-CD3 (5 mg/ml, clone OKT3) and anti-CD28 (5 mg/ml, clone PV-1) (BioXcell), to a final concentration of 3×10⁵ splenocytes/200μl/well. 72h later, supernatants were harvested and assayed for IFNγ production by ELISA (R&D Systems).

Steinberg S.M., Zhang P., Malik1 B.T., Boni A., Shabaneh T.B., Byrne K.T., Mullins D.W., Brinckerhoff C.E., Ernstoff M.S., Bosenberg M, & Turk M.J. (2014). BRAF-inhibition alleviates immune suppression in murine autochthonous melanoma. Cancer immunology research, 2(11), 1044-1050.

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Activated T Cell Phenotyping by Flow Cytometry

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Flow cytometry was performed on a FACS LSR II or Fortessa (BD Biosciences). For lymphocyte activation studies, soluble anti-CD3 (500 ng/ml; clone OKT3, Bio X Cell) ± anti-CD28 (1 μg/ml; clone CD28.2, BD Biosciences) or IL-2 (PeproTech) was added to media for 2 days. Proliferation was measured by CFSE (eFluor 670, BD Biosciences) after 3 days. EVs were added every 24 hours (5 μg/ml). PD1 blockade was achieved by adding anti-PD1 (10 μg/ml; EH12) or immunoglobulin G control (clone MOPC-21, Bio X Cell). Cells were gated by FSC (forward scatter)/SCC (side scatter) while excluding duplets by FSC-A/FSC-H. Subsequently, CD3⁺CD56⁻ T cells were gated with further classification of CD4⁺ and CD8⁺ T cells. Finally, CD4⁺ and CD8⁺ T cells were measured for their CD69, CD25, PD1, and TIM3 expression (seen as a downloadable figure). Cells were stained with the following antibodies: CD3/PE-Cy5.5 (1:100; clone SK7, eBioscience), CD4/FITC (1:100; clone RPA-T4, BD Biosciences), CD8/AmCyan (1:10; clone SK1, BD Biosciences), CD16/APC-Cy7 (1:100, BD Biosciences), CD25/PE-Cy7 (1:10; clone BC96, eBioscience), CD56/V450 (1:30; clone B159, BD Biosciences), CD69/APC (1:10; clone FN50, BD Biosciences), TIM3/APC-Cy7 (1:100, clone F38-2E2, BD Biosciences), and PD1/PE (1:100; clone J105, eBioscience).

Ricklefs F.L., Alayo Q., Krenzlin H., Mahmoud A.B., Speranza M.C., Nakashima H., Hayes J.L., Lee K., Balaj L., Passaro C., Rooj A.K., Krasemann S., Carter B.S., Chen C.C., Steed T., Treiber J., Rodig S., Yang K., Nakano I., Lee H., Weissleder R., Breakefield X.O., Godlewski J., Westphal M., Lamszus K., Freeman G.J., Bronisz A., Lawler S.E, & Chiocca E.A. (2018). Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Science Advances, 4(3), eaar2766.

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Induction of Regulatory T Cells from PBMCs

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PBMCs from freshly drawn heparinized blood originating from healthy volunteers were isolated by density gradient centrifugation using Ficoll Paque (GE). PBMCs (1 × 10⁵/well) were incubated for 96 h in the presence of DMSO or Cpd A (1 μM) together with anti-CD3 (3 ng/ml, clone OKT-3, BioXcell), anti-CD28 (30 ng/ml, clone CD28.2, BioLegend), TGF-β1 (0.2 ng/ml, Biolegend), IL-2 (15 ng/ml, Biolegend), anti-IFN-γ antibody (5 μg/ml, Biolegend), and anti-IL-4 (5 μg/ml, Biolegend). Cells were prepared for CD4/CD25 surface staining, followed by intracellular FoxP3 staining.

Ecoeur F., Weiss J., Kaupmann K., Hintermann S., Orain D, & Guntermann C. (2019). Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses. Frontiers in Immunology, 10, 577.

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RORγt Modulates IL-17A Production

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HUT78 T-cells stably expressing full-length eGFP-RORγt or eGFP-empty vector were stimulated and Cpd A-induced inhibition of IL-17A production was measured as described before (45 (link)). Briefly, HUT78 cells stably expressing RORγt (5 × 10⁴/well) were stimulated with PMA (8 ng/ml, Sigma) plus CD3 mAb (2.5 μg/ml, clone OKT-3, BioXcell) and incubated in X-vivo 15 medium containing 10% FCS with diluted Cpd A. After 48 h of incubation at 37°C, the supernatants were collected and IL-17A or IL-2 were quantified by ELISA according to the manufacturer‘s specifications (eBioscience or Biolegend).

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Murine Splenocyte T-cell Activation Assay

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Tumors were digested as above, and CD11b⁺ cells were isolated magnetically by positive selection (Miltenyi). Purified CD11b⁺ cells were combined at indicated ratios with RBC-depleted C57BL/6 mouse splenocytes, and added to a 96 well plate pre-coated with anti-CD3 (5mg/ml, clone OKT3) and anti-CD28 (5mg/ml, clone PV-1) (BioXcell); to a final concentration of 3×10⁵ splenocytes/200µl/well. Seventy-two hours later, supernatants were harvested and assayed for IFN-γ production by ELISA (R&D Systems).

Steinberg S.M., Shabaneh T.B., Zhang P., Martyanov V., Li Z., Malik B.T., Wood T.A., Boni A., Molodtsov A., Angeles C.V., Curiel T.J., Whitfield M.L, & Turk M.J. (2017). Myeloid cells that impair immunotherapy are restored in melanomas which acquire resistance to BRAF inhibitors. Cancer research, 77(7), 1599-1610.

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