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Anti cd3 mab

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The Anti-CD3 mAb is a monoclonal antibody that binds to the CD3 complex, which is a part of the T cell receptor (TCR) complex on the surface of T cells. This antibody can be used for the detection and analysis of T cells in various applications.

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12 protocols using anti cd3 mab

1

Neoepitope-specific CD8+ T cell expansion

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Putative neoepitope HLA class I multimers were generated using conditional MHC class I ligands and peptide exchange technology [12 (link)]. Healthy donor PBMCs isolated from anonymised National Blood Service apheresis cones were incubated with phycoerythrin (PE)-labelled neoepitope multimers, before enrichment by magnetic-activated cell sorting (MACS) using anti-PE microbeads [Miltenyi Biotec]. Enriched cells were expanded using anti-CD3 mAb (30 ng/ml) [ebioscience] and IL-2 (3000 U/ml) [Novartis]. Neoepitope multimer+ CD8+ T cell populations were identified by combinatorial-encoded multimer staining [13 (link)]. To establish T cell clones, neoepitope multimer+ CD8+ cells were single-cell sorted and expanded using anti-CD3 mAb (30 ng/ml) [ebioscience] and IL-2 (3000 U/ml) [Novartis]. T cell clones were confirmed by neoepitope multimer and anti-CD8-APC (1:50) [BD] staining.
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2

Expansion of Antigen-Specific T Cells

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Peripheral mononuclear blood cells (PBMCs) were isolated using Ficoll density gradient centrifugation from healthy donors supplied by the Beijing Blood Bank. PBMCs were cultured at 1×106/ml in RPMI-1640 medium supplemented with 10% FBS and 5 μg/ml anti-CD3 mAb (eBioscience, San Diego, CA, USA) and 100 IU/ml recombinant human IL-2 at 1×106/ml. Fresh medium containing 100 IU/ml recombinant human IL-2 was added to cell culture as the method previously described [27 (link), 28 (link), 36 (link)]. On day 13, ATC expansion products of donors averaged 97.33 ± 0.86% CD3+ cells (32.53± 3.00% CD4+ and 64.87 ± 0.81% CD8+) were used immediately or cryopresered for further use.
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3

PBMC Activation and Expansion for ATC Generation

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Peripheral blood mononuclear cells (PBMCs) were separated immediately by Ficoll-Hypaque density gradient centrifugation. Blood was obtained from healthy donors which supplied by the Beijing Blood Bank. PBMCs were cultured at 1×106/ ml in RPMI-1640 medium supplemented with 10% FBS and 5 μg/ml anti-CD3 mAb (eBioscience, San Diego, CA, USA) combined with 5 μg/ml anti-CD28 mAb (eBioscience) in the presence of 100 IU/ml recombinant human IL-2 (Peprotech, Rocky Hill, NJ, USA). IL-2 was added every 2 or 3 days to stimulate ATC cells and the cells were cultured for 14 days. On day 14, ATC expansion products of healthy donors were on 98.99% CD3+ cells (2.66% CD3+CD4+ cells, 85.95% CD3+CD8+ T cells, and 16.16% CD3+CD56+T cells), the cells were used immediately or cryopreserved for further use.
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4

Isolation and Differentiation of CD4+ T Cells

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Peripheral blood cells were collected and isolated with Ficoll-PaqueTM (GE healthcare, Uppsala, Sweden) as described previously.19 (link) Briefly, peripheral blood (3 mL) diluted with sterile PBS (3 mL) was added to equal volume Ficoll. The tubes were vortexed for 30 minutes under 2,000 rpm at 20°C. PBMCs were collected and incubated with CD4+ microbeads for 30 minutes at 4°C. CD4+ T-cell clones were expanded after being stimulated with plate-bound anti-CD3 mAb (5 mg/mL), and anti-CD28 mAb (2 mg/mL, eBioscience, San Diego, CA), followed by being stimulated with recombined human (rh) TGFβ (20 ng/mL), rh IL-6 (30 ng/mL), anti-IFNγ (20 µg/mL), and anti-IL-4 (20 µg/mL) with PBS or TWP (1.25 mg/mL) for 72 hours. Differentiated CD4+ T-cells were collected for qRT-PCR. CD4+ T-cells were stimulated with mentioned cocktails for 5 days followed by being stimulated with PMA (50 ng/mL, Sigma-Aldrich, St Louis, MO, USA), ionomycin (750 ng/mL, Sigma-Aldrich), and Brefeldin A (3  μg/mL, eBioscience) for 5 hours. Differentiated CD4+ T-cells were collected for cell membrane and intracellular staining, eg, CD25, IFNγ, IL-17A, and FOXP3 for flow cytometer analysis.
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5

Treg-Mediated Suppression of T-Cell Proliferation

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CD4+CD25 T cells (Teff) and CD4+CD25+ T cells (Tregs) were cocultured in 96-well plates coated with 50 ng/mL anti-CD3 mAb (eBioscience, USA) at a density of 104 cells/well with different Teff/Treg ratios (1 : 1, 1 : 1/2, 1 : 1/4, and 1 : 1/8). All wells were cultured in a final volume of 200 μL with the presence of T cell-depleted and irradiated antigen presenting cells (105 cells/well). After 72 h, [3H]-thymidine (1 μCi/well) was added for 16 h prior to the determination of proliferation by scintillation counting (MicroBeta1450 Liquid Scintillation Counter; Perkin Elmer, USA). Percent inhibition of proliferation was determined as follows: (1-[3H]-thymidine uptake of cocultured Treg and Teff)/Teff alone × 100%. Triplicate wells were used in all suppression experiments.
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6

Analyzing Immune Cell Subsets by Flow Cytometry

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Icariin was purchased from MEYER company (Shanghai, China), whereas the antibodies used for western blotting were purchased from Cell Signaling Technology (Danvers, MA). The antibodies were anti-CD3 mAb (PE), anti-CD4 mAb (APC), anti-CD8a mAb (PE/Cy7), anti-CD25 mAb (APC/Cy7), anti-CD44 mAb (FITC), anti-CD62L mAb (APC/Cy7), anti-IFN-γ mAb (PE), HO-1 mAb (PE/Cy7), anti-CD16/32 mAb and anti-Foxp3 mAb (FITC) (eBioscience, San Jose, CA, USA).
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7

T Cell Activation and Cytokine Production

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Ninety-six well tissue culture plates were prepared by adding 1 µg/ml anti-CD3 mAb (OKT3- eBioscience), and/or 2 µg/ml recombinant human PD-L1 and coated overnight at 4 °C. After washing the wells with PBS, 100,000 CD3+ cells isolated by magnetic bead separation from peripheral blood mononuclear cells (PBMC) were added to each well in media. The plates were incubated for 3 days at 37 °C and 5% CO2, and supernatants were harvested and analyzed for IL-2 or IFN-γ production by ELISA (R&D Systems).
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8

Expansion and Characterization of Activated T Cells

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Peripheral blood mononuclear cells (PBMCs) were separated immediately by Ficoll‐Hypaque density gradient centrifugation. Blood was obtained from healthy people which provided by the Beijing Blood Bank. PBMCs were cultured at 1 × 106/mL in RPMI‐1640 medium with 10% FBS. The ATC cells were stimulated by 5 μg/mL anti‐CD3 mAb (eBioscience, San Diego, CA, USA) and interleukin‐2 every. On day 13, the amplified ATC of healthy donors were 98.34% CD3+ cells (9.38% CD3+ CD4+ cells and 87.9% CD3+ CD8+ T cells), and some CD3+ cells co‐expressed CD56+ (23.57% CD3+ CD56+ T cells) which were used or cryopreserved.
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9

Naive CD4+ T Cell Polarization by IL-33

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Liver naïve CD4+ T cells (CD4+ CD25 CD44 CD62L+) were sorted from the infected WT mice as previously described.31 (link) Naïve CD4+ T cells were then cultured (3×105 cells/well) in 96-well plates coated with anti-CD3 mAb (5 μg/mL, eBioscience) and soluble anti-CD28 mAb (3 μg/mL, eBioscience) and kept in the presence of IL-2 (100 U/mL, eBioscience). Exogenous rmIL-33 (10 ng/mL) was added to investigate its ability to polarize Treg and PBS used as the control. Cells were cultured in RPMI-1640 Medium, 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL of Penicillin/Streptomycin for 5 days.
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10

Flow Cytometry Analysis of BMNC

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BMNC isolated as described28 were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD45, anti-F4/80, anti-Gr-1 or appropriate isotype control mAb (all from eBiosciences). CFSE (0.7 mg in 0.3 ml PBS-DMSO 7%; Santa Cruz Biotechnologies) or vehicle were injected i.v. on day 5 post-infection as reported3 (link). BMNC were then collected and stained with anti-CD11b, anti-CD11c or isotype control mAb (eBiosciences) and examined for expression of CFSE. Splenocytes were incubated with or without anti-CD3 mAb and Brefeldin A (10 μg/ml; eBiosciences). Cells were stained with anti-CD3, anti-CD4 and anti-CD8 (eBiosciences), permeabilized, stained with anti-IFN-γ or isotype control mAb (eBiosciences) and analyzed in an LSR II flow cytometer (BD Biosciences).
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