The cells were labeled with the CellTrace™ CFSE Cell Proliferation Kit (cat. no. 65-0850-84; ThermoFisher Scientific, Inc.) according to the manufacturer’s protocols. After labeling, the CAR-T cells were co-cultured with Aspc-1 tumor cells at a 1:1 ratio for 2 days at 37°C, followed by staining of the cells with an anti-human CD3 antibody (Biolegend, 300311) and analyses by Flow cytometry.
Anti human cd3 antibody
Manufactured by BioLegend
Sourced in United States
The Anti-human CD3 antibody is a laboratory reagent used for the detection and investigation of human CD3 protein, a key component of the T-cell receptor complex. This antibody can be utilized in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and analyze T-cells.
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Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti human cd28 antibody, by BioLegend (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Eurobio Scientific (1 mentions)
Anti human cd25 antibody, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
2 mercaptoethanol 2 me, by Thermo Fisher Scientific (1 mentions)
Anti vegfr2 antibody, by Abcam (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
Golgiplug protein transport inhibitor, by BD (1 mentions)
Anti vegf r1 antibody, by R&D Systems (1 mentions)
Vegf a, by MedChemExpress (1 mentions)
Mk 2206 2hcl, by MedChemExpress (1 mentions)
Vegf a, by Selleck Chemicals (1 mentions)
Vegf a, by R&D Systems (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Attune acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Human il 2, by Cytek Biosciences (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
15 protocols using anti human cd3 antibody
1
Cell Proliferation Assay for CAR-T Cells
2
Multiparameter flow cytometry analysis of T cell activation and B cell HLA-DR expression
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For analyzing DR2a and DR2b expression levels on B cells and monocytes, PBMCs from HLA-DR15+ donors were incubated with human IgG (Sigma-Aldrich) to block unspecific antibody binding to Fc-receptors, labeled with Live/Dead® Aqua (Invitrogen), and then stained for surface marker using fluorochrome-conjugated antibodies, including Alexa Fluor 488-conjugated anti-DR2a antibody (5 μg/ml, One Lambda, Thermo Fisher Scientific) and Alexa Fluor 647-conjugated anti-DR2b antibody (5 μg/ml, One Lambda, Thermo Fisher Scientific), at 4°C. For analyzing CD69, CD25, and TCR α/β expression on TCCs after stimulation, TCCs were harvested at different time points after stimulating with peptides and washed twice with PBS. TCCs were then incubated with human IgG (Sigma-Aldrich), labeled with Live/Dead® Aqua (Invitrogen), and stained for surface marker using fluorochrome-conjugated antibodies, including anti-human CD69 antibody, anti-human CD25 antibody, anti-human TCR α/β antibody, anti-human CD4 antibody, and anti-human CD3 antibody (Biolegend; Key Resources Table ), at 4°C. Cells were washed twice after staining and resuspended with cold PBS containing 2mM ethylenediamine tetraacetic acid (EDTA, AppliChem) and 2% FCS (Eurobio). Measurement was performed using LSR Fortessa Flow Cytometer (BD Biosciences) and data were analyzed using FlowJo (Tree Star).
3
Modulation of CD4+ T Cell Function by VEGF-A
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For primary CD4+ T-cell isolation, PBMCs were acquired utilizing Ficoll Paque Plus (GE Healthcare, USA) gradient cell separation according to the manufacturer’s instructions. Next, the CD4+ T Cell Isolation Kit, human (Miltenyi Biotec, Germany) was utilized to isolate CD4+ T cells. Then, the cells were resuspended at 1 × 106 cells/mL and cultured in advanced RPMI1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% 2-mercaptoethanol (2-ME; Gibco) and 1% penicillin–streptomycin (Gibco) in 48-well plates precoated with 2 μg/mL anti-human CD3 antibody (BioLegend, USA). For VEGF-R-related assays, after incubation with 5 μg/mL anti-VEGF-R1 antibody (R&D Systems, USA) and/or 10 μg/mL anti-VEGF-R2 antibody (Abcam, USA) for 1 h, CD4+ T cells were treated with blank (CON) or 15 ng/mL VEGF-A (R&D Systems, USA) for 2 days respectively. GolgiPlug Protein Transport Inhibitor (BD Bioscience, USA) was administered for the last 6 h to detect the cytotoxic molecules. Regarding AKT/mTOR inhibition assays, CD4+ T cells were treated with blank (CON), 15 ng/mL VEGF-A, VEGF-A + 10 μM MK-2206 2HCl (AKT inhibitor, MedChemExpress, USA), VEGF-A + 100 nM rapamycin (mTOR inhibitor, Selleck, China) or VEGF-A + 10 μM MK-2206 2HCl + 10 μM MHY1485 (mTOR activator, MedChemExpress, USA) for 2 days in the presence of GolgiPlug Protein Transport Inhibitor for the last 6 h.
4
Isolation and Characterization of CRTAM+ T Cells
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PBMCs were isolated from healthy donors by centrifugation over Ficoll density gradient. CD4+ T cells were isolated by anti–human CD4 MACS beads (Miltenyi Biotec) and were stimulated by 10 µg/ml of anti–human CD3 antibody (OKT3) for 14 h. After stimulation, T cells were stained with anti–human CRTAM mAb (Cr24.1; BioLegend), and CRTAM+ and CRTAM− cells were sorted by FACSAria and incubated with 2,000 U/ml of human IL-2 (Ajinomoto) for 5 d. These experiments were performed in compliance with the institutional guidelines of the Tokyo University of Science (Tokyo, Japan), and all subjects provided informed consent as approved by the ethical committee. Healthy volunteers were recruited after obtaining informed consent.
5
Leukocyte Subset Purity Validation
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To confirm cell purity, isolated leukocyte subsets were analyzed by flow cytometry with an Attune® Acoustic Focusing Cytometer (Applied Biosystems) by accessing their dispersion pattern or after staining with specific cell surface markers, such as anti-human CD3 antibody (BioLegend, San Diego, CA, USA) to stain T lymphocytes, and anti-human CD16 antibody (BD Biosciences, San Jose, CA, USA) to stain neutrophils.
6
PBMC Isolation and T Cell Activation
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Human PBMC were isolated from fresh buffy coat collected from healthy donors using the density gradient centrifugation method. Briefly, buffy coat was diluted with 2X volume PBS and was gently layered on top of an equal volume Ficoll-Paque PLUS (#17144003, Cytiva, Marlborough, MA), followed by centrifugation at 400 x g for 40 min without break. The PBMC were removed and transferred to a new 50 ml tube, washed in 25 ml DPBS buffer twice, and recovered by centrifugation at 350 x g for 10 min. The cells were counted and cultured in the presence of 1 µg/ml anti-human CD3 antibody (#317302, BioLegend, San Diego, CA) and 2 µg/ml anti-human CD28 antibody (#302902, BioLegend) for 72 h. The cells were then infected with NL4-3 at a MOI as indicated in the presence of 8 µg/ml polybrene by spinoculation at 850 x g, room temperature for 2 h. The cells were recovered by centrifugation, washed with fresh media, and continued to culture in the presence of 100 IU/ml human IL-2 (#21-8029-U050, Tonbo Biosciences, San Diego, CA) and used in subsequent Metformin experiments.
7
Isolation and Activation of T Cells from SLE Patients
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Peripheral blood mononuclear cells (PBMC) were isolated from SLE patients and healthy donors using Ficoll (GE Healthcare, Amersham, UK) gradient centrifugation. A part of the PBMCs was stimulated with 1 μg/mL phytohaemagglutinin-L (PHA-L, Sigma-Aldrich, St. Louis, MO, USA) and the cells were cultured for 72 h in a humidified incubator with 5% CO2 at 37 °C in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Paisley, UK), 2 mM L-glutamine (Gibco, Life Technologies, Paisley, UK) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA), henceforward referred to as activated T cells. Activated T cell cultures were > 90% pure, as controlled with flow cytometry using anti-human CD3 antibody (BioLegend, San Diego, CA, USA) (data not shown).
8
Isolation and Culture of CD8+ T Cells
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Isolation of peripheral blood mononuclear cells (PBMCs) from the peripheral blood of the patients was carried out according to a standard protocol using lymphocyte separation medium (MP Biomedicals, Irvine, CA, SKU 0850494-CF). Then, PBMCs were cultured with 200U IL-2, and two days later, CD8+ T cells were isolated by using human CD8 Microbeads (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, 130-045-201, Bergisch Gladbach, Germany), LS column (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, 130-042-401), and MidiMACS Separator (130-042-302). RPMI containing 10% human male AB plasma (Sigma-Aldrich, MO, USA, H4522), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, 1% penicillin/streptomycin, 10 mM HEPES (Life Technologies, Waltham, MA, USA), 200 IU/mL recombinant human IL-2 (200-02-500UG, PeproTech, Cranbury, NJ, USA), and 50 ng/mL antihuman CD3 Antibody (BioLegend, San Diego, CA, USA, 317302) were used for the first 48 h of culture. Then, culturing and passaging was done using complete media without antihuman CD3 Antibody.
9
Efficient Murine and Human T Cell Isolation
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Murine T cells were enriched from spleens using the EasySep CD8 T cell enrichment kit (StemCell Technologies) and cultured in RPMI-1640 media supplemented with 10% FBS, 1% penicillin–streptomycin, 1% GlutaMAX, 10 mM HEPES, 1 mM sodium pyruvate, and 55 μM 2-mercaptoethanol. B16-F10 (ATCC) and MC38 (obtained from Dr. Wucherpfennig’s lab) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The media for ovalbumin-expressing B16 cells (obtained from Dr. Wucherpfennig’s lab) was further supplemented with 0.5 mg mL−1 geneticin. All cell lines were tested for mycoplasma and confirmed to be negative. All supplements were obtained from Life Technologies. Blood collars for human T cells were obtained from the Brigham and Women’s Hospital Blood Donor Center. Appropriate consent was obtained from all donors. T cells were enriched using the Rosette Sep Human T cell enrichment kit, and cells were separated via ficoll gradient separation using SepMate. T cells were cultured in ImmunoCult-XF T Cell Expansion Medium supplemented with 10 ng mL−1 IL-2 (Peprotech) and activated with 25 µl mL−1 ImmunoCult Human T Cell Activator (all from StemCell Technologies). The purity of the isolated cells was determined using anti-human CD3 antibody (BioLegend) and confirmed to be greater than 95% purity.
10
Investigating Immune Checkpoint Modulation in Melanoma
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Human melanoma A375 cell line (CRL-1619) and TALL-104 T lymphoblast cell line (CRL-11386) were purchased from ATCC and maintained according to the protocols provided by the vendor. TALL-104 cells were first stimulated with anti-human CD3 antibody (5µg/ml) (Biolegend) and anti-human CD28 antibody (5µg/ml) (#MABF408, Millipore). Then, the activated TALL-104 cells and A375 melanoma cells were resuspended together at a 6:1 ratio in complete RPMI media, dispensed into the multi-well slide chambers and incubated for 1 hour in 5% CO2 at 37˚C then isotype control or testing antibodies were added to the co-cultured cells. The effects of the isotype control antibodies were compared to the effects of antibodies against CHI3L1 (FRG; 5μg), CTLA-4 (5μg), FRG+CTLA-4 administered simultaneously (2.5μg each) and FRGxCTLA-4 (5μg). After 48 hours of incubation, cells were subjected to TUNEL and immunocytochemistry evaluations using fluorescent-labeled antibodies against CD8, perforin, granzyme-B and PTEN by as previously described (30 (link)).
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