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Apc conjugated anti human cd3

Manufactured by BioLegend
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APC-conjugated anti-human CD3 is a monoclonal antibody that recognizes the CD3 complex on human T cells. CD3 is a T cell co-receptor that is part of the T cell receptor (TCR) complex and plays a crucial role in T cell activation and signaling. The APC (Allophycocyanin) fluorochrome conjugated to the anti-CD3 antibody allows for the identification and analysis of T cells by flow cytometry.

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9 protocols using apc conjugated anti human cd3

1

T Cell Purity Validation Using Flow Cytometry

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To check the purity of isolated T cells using magnetic beads, isolated T cells were stained with APC-conjugated anti‐human CD3 (Clone: UCHT1, BioLegend). Mesothelin expression was detected using PE-conjugated anti-human mesothelin (Clone: #420411, R&D Systems). All samples were acquired with a BD FACSCalibur (BD Biosciences) and analyzed using FlowJo software (v10.6). All assays were performed in duplicate and repeated two to three times.
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2

Characterization of Activated T Cell Phenotype

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The purity of isolated T cells was confirmed using APC-conjugated anti-human CD3 (Clone: UCHT1, BioLegend). PE-conjugated anti-human mesothelin (Clone: #420411, R&D Systems) was used to detect mesothelin expression. FITC-conjugated anti-human CD3 (Clone: HIT3a, BioLegend), PE-conjugated anti-human CD279 (PD-1) (Clone: EH12.2H7, BioLegend), and APC-conjugated anti-human CD366 (Tim-3) (Clone: F38-2E2, BioLegend) antibodies were used to measure the expression of exhaustion markers. For proliferation assays, cells were loaded with CellTrace™ CFSE (Life Technologies, #C34554) according to manufacturer’s instructions, and T cells were detected using PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend). Data were collected using a BD FACSCalibur (BD Biosciences) and analyzed with FlowJo software (v10.6). All assays were performed in duplicate and repeated two to three times.
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3

Lentiviral Transduction of Activated T Cells

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Buffy coats or fresh whole blood from healthy donors were purchased from the Iranian Blood Transfusion Organization (IBTO) and handled with necessary safety procedures and ethical requirements. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Paque gradient centrifugation. T cells were negatively selected from the PBMCs using immunomagnetic beads (pan T-cell isolation kit II, human, Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of isolated cells was assayed using APC conjugated anti-human CD3 (BioLegend, San Diego, CA). T cells were then activated by anti-CD3/CD28 antibody-coated beads (Cell/Bead ratio: 1/3) (Life Technologies, Grand Island, NY). After 3 days, the anti CD3/CD28 beads were removed and activated T cells were transduced by appropriate amount of concentrated lentiviral supernatants (at multiplicity of infection [MOI] of 7) supplemented with 0.8 μL of polybrene (Santa Cruz Biotechnology, Santa Cruz, CA), then plate was spinofected at 2100 rpm at 32 °C for 1 h, followed by incubation at 37 °C. Six hours later, 1 ml of fresh media containing 100 IU IL-2 (Miltenyi Biotec) was added to each well. After 4 days, CAR expressing cells were detected using goat anti-human IgG F (ab’)2 Biotin (BioRad, Hercules, CA)- Streptavidin APC (BioRad).
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4

Isolation and Activation of Human T Cells

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Whole blood was taken from healthy donors. Using Ficoll–Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5 × 106 cells/well) and cultured in RPMI1640 containing 10%FBS and 100 IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37 °C, the purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA) by flow cytometry. The clone of APC anti-human CD3 Antibody was UCHT1.The purity of T cells is represented in the figure S 2.
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5

Multiparametric Flow Cytometry Assay

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Brilliant Violet 421-conjugated anti-human CD45 (HI30), APC-conjugated anti-human CD3 (OKT3), APC-conjugated anti-human CD19 (HIB19), FITC-conjugated anti-human CD14 (M5E2), CD11c-conjugated anti-human CD11c (3.9), PE-Cy7-conjugated anti-human HLA-DR (LN3), PerCP-Cy5.5-conjugated anti-human CD56 (5.1H11), PE-conjugated anti-human LILRB4 (ZM4.1), Pacific blue-conjugated anti-mouse CD45 (30-F11), Brilliant violet 510-conjugated anti-mouse CD11b (M1/70), PerCP-Cy5.5-conjugated anti-mouse CD11c (N418), FITC-conjugated anti-mouse Ly6G (1A8), PE-Cy7-conjugated anti-mouse CD64 (X54-5/7.1), APC/Fire 750-conjugated anti-mouse CD24 (M1/69), APC-conjugated anti-mouse IA/IE (M5/114.15.2), PE-conjugated anti-mouse LILRB4 (H1.1), PE-conjugated American Hamster IgG control antibody (HKT888) were purchased from Biolegend. APC conjugated anti-human CD206 (19.2), APC-conjugated mouse IgG1κ control antibody (P3.6.2.8.1), PE-conjugated mouse IgG1κ control antibody (P3.6.2.8.1) were purchased from eBiosciences.
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6

Characterizing PBMC Activation by hADSC Secretome

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PBMCs were plated in 35 mm non-treated dishes (2 × 106 cells per dish) in 2 mL of CM harvested from hADSCs-IL2, native hADSCs or fresh DMEM/F12. Activation of PBMCs populations was determined at 72 h by flow cytometry using FACS Aria III (BD Biosciences, San Jose, CA, USA), a minimum of 20,000 events were acquired for each sample. The following populations of the immune cells were investigated: late-activated T-cells (CD3+, HLA-DR+), activated CD8+ T-cells (CD8+, CD38+), early-activated T-cells (CD3+, CD25+), NK cells (CD3, CD56+), NKT-cells (CD3+, CD56+). PBMCs were washed twice with PBS and resuspended at 1 × 105 cells/mL in PBS before being incubated with antibodies for 30 min in the dark at RT. All samples were stained with allophycocyanin (APC)-conjugated anti-human CD3 (#300312, BioLegend, San Diego, CA, USA) or Alexa Fluor 488-conjugated anti-human CD3 (#2324020, Sony Biotechnology, San Jose, CA, USA), Alexa Fluor 488-conjugated HLA-DR (#IM0463U, Beckman Coulter, USA), Alexa Fluor 647-conjugated anti-human CD56 (NCAM) (#2191564, BioLegend, San Diego, CA, USA), R-phycoerythrin (PE)-Cy7-conjugated anti-human CD25 (#2113060, Sony Biotechnology, San Jose, CA, USA), PE-conjugated anti-human CD8a (#300908, BioLegend, San Diego, CA, USA) and Alexa Fluor 488-conjugated anti-human CD38 (#2117515, Sony Biotechnology, San Jose, CA, USA).
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7

Isolation and Activation of Human T Cells

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Whole blood was taken from healthy donors. Using Ficoll-Paque density gradient centrifugation, human peripheral blood mononuclear cells (PBMCs) were isolated. Isolated PBMCs were seeded into 24-well plates (1.5×10 6 cells/well) and cultured in RPMI1640 containing 10%FBS and 100IU hIL-2 (Miltenyi Biotec). To activate and enrich T cells, PBMCs were cultured with 3 ug/ml anti-CD3 antibody (Miltenyi Biotec) and 10 ug/ml anti-CD28 antibody (Miltenyi Biotec). After 4 days of incubation at 37°C purity of T cells was assayed using APC conjugated anti-human CD3 (BioLegend, USA).
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8

Detecting Antigen-Specific T Cell Responses

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T cells recovered from lymph nodes (LNs) were also analyzed by intracellular staining for detection of antigen-specific IFN-g secretion, as described. 4 As a negative control for gating, nonstimulated lymphocytes were used. T cells expanded homeostatically and antigenically were stimulated for 16 hours with 10 mg/mL of CMV PepTivator (pp65 overlapping peptide pool; Miltenyi Biotec). The Wilms tumor 1 (WT1) overlapping peptide pool (Miltenyi Biotec) was used as negative control for peptide stimulation. Protein transport inhibitor cocktail (eBioscience, Frankfurt, Germany) was added to the cells 2 hours after stimulation. After 16 hours, T cells were harvested, stained with APC-conjugated anti-human CD3, APC-H7 conjugated CD4, and PCy7-conjugated anti-human CD8 antibodies (Biolegend). After fixation/permeabilization with Cyofix/perm (BD Biosciences) for 20 minutes at 4 C and washing, anti-human phosphatidylethanolamineeIFN-g (eBioscience) was used for staining for 30 minutes. The cells were acquired by flow cytometry using LSRII (BD Biosciences) and analyzed by FlowJo software.
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9

Flow Cytometry Staining Protocol

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PE-conjugated anti-human PD-L1, human IgG isotype control antibodies, PE-conjugated anti-human CD69, APC-conjugated anti-human CD3 and PE-conjugated anti-human CD8 antibodies were purchased from Biolegend. Cells were assayed using a ow cytometer, and data were processed using the accompanying software (CytExpert, Beckman Coulter, Beijing,China).
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