The following mAbs were used for phenotypic analysis: APC-Cy7 Mouse Anti-Human CD3; FITC-anti-human CD4; APC-anti-human CD8; (BD Biosciences). Tumor cell surface expression of HER2 was detected by trastuzumab antibody (Herceptin, Genentech, San Francisco, CA), CD20 by biotinylated rituximab (Rituxan; Genentech, South San Francisco, CA, USA) followed by incubation with Strepavidin-APC. BsAb-IR expression was detected by Mov18/ZEL antibody (Enzo Life Sciences) followed by anti-mouse FITC or APC conjugated antibody (LifeBioscience). The isotype controls mIgG-APC-Cy7, mIgG-FIT, mIgG-APC, mIgG-PerCpCy5, mIgG-Pacific-Blue-A, and mIgG-PE-Cy7 were from eBiosciences and anti-mouse-AF488 from Invitrogen. Flow cytometric data were analyzed by FlowJo software. For intracellular staining, cells were permeablized using the FoxP3 staining buffer set (eBioscience), according to manufacturer’s instructions and labeled with: anti-human IFNg-PacificBlue, anti-human IFNg-PerCpCy5, anti-human TNFa-APC, anti-human IL2-PeCy7 (BD Biosciences).
Apc cy7 mouse anti human cd3
Manufactured by BD
Sourced in United States, United Kingdom
The APC-Cy7 Mouse Anti-Human CD3 is a fluorescence-conjugated monoclonal antibody designed for the identification and enumeration of human T cells by flow cytometry. It binds to the CD3 cell surface antigen, which is expressed on mature T lymphocytes.
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Lab products found in correlation
Anti human cd4 fitc, by BD (1 mentions)
Apc anti human cd8, by BD (1 mentions)
Rituxan, by Genentech (1 mentions)
Anti mouse af488, by Thermo Fisher Scientific (1 mentions)
Mov18 zel antibody, by Enzo Life Sciences (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Flex t hla a 02 01 monomer uvx, by BioLegend (1 mentions)
Alexa fluor 700 mouse anti human cd8, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Streptavidin allophycocyanin apc, by BioLegend (1 mentions)
Brilliant violet 421 anti human cd197, by BioLegend (1 mentions)
Apc mouse anti human cd4, by BD (1 mentions)
Fluorescein isothiocyanate fitc anti human cd45ra, by BioLegend (1 mentions)
Pe mouse anti human cd2, by BD (1 mentions)
Cd127 pe cy7, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti human foxp3, by BioLegend (1 mentions)
Percp cy5.5 mouse anti human cd4, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Fitc mouse anti human cd4, by BD (1 mentions)
Pharm lyse lysing buffer, by BD (1 mentions)
5 protocols using apc cy7 mouse anti human cd3
1
Multicolor Flow Cytometry for Phenotypic Analysis
2
Multi-color FACS Analysis and Sorting
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Cells were collected, washed, and resuspended in FACS buffer, and stained with fluorescent dye conjugated antibodies for 15 minutes at 4 °C. Peptide-MHC tetramers were generated through ultaviolet-irradiation-mediated peptide exchange method31 (link). APC-Cy™7 Mouse Anti-Human CD3 (BD Biosciences, 557832, 2.5 μl/5 × 105 cells), Alexa Fluor® 700 Mouse Anti-Human CD8 (BD Biosciences, 557945, 2.5 μl/5 × 105 cells), and Flex-T™ HLA-A*02:01 Monomer UVX (BioLegend, Cat#280004, 0.05 μg/1 × 106 cells) were used. After washing twice in FACS buffer, cells were analyzed using a FACSAria II (BD Biosciences) with live cell gating based on 4’,6-diamidino-2-phenylindole (DAPI) exclusion, and CD8+ pMHC-tetramer+ cells were sorted. The data were analyzed using FlowJo software (Tree Star).
3
Comprehensive Immunophenotyping of CAR-T Cells
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Flow cytometry cell staining was performed at 4°C in PBS supplemented with 2% FBS, unless otherwise indicated. The expression of MSLN on cancer cells was detected by flow cytometry, using a human MSLN PE-conjugated antibody (R&D, FAB32652P, USA).
The expression of CAR on CAR-T cells was detected using biotinylated human MSLN (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
The immunophenotypes of T cells were tested using flow cytometry. The antibodies used for analysis include: APC-Cy 7 mouse anti-human CD3 (BD, NJ, USA), APC mouse anti-human CD4 (BD, NJ, USA), phycoerythrin (PE) mouse anti-human CD8 (BD, Franklin Lakes, NJ, USA), Brilliant Violet 421 anti-human CD197 (BioLegend, CA, USA), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, San Diego, CA, US), PE-Cy7 anti-human CD45RO (BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD197 (CCR7; BioLegend, San Diego, CA, USA), Brilliant Violet 510 anti-human CD62L (BioLegend, San Diego, CA, USA), Brilliant Violet 605 anti-human CD95 (Fas; BioLegend, San Diego, CA, USA), PE anti-human CD223 (LAG-3; BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD366 (Tim-3; BioLegend, San Diego, CA, USA), and APC anti-human CD279 (PD-1; BioLegend, San Diego, CA, USA). Data were analyzed using NovaExpr software (ACEA, Ashland, OR, USA).
The expression of CAR on CAR-T cells was detected using biotinylated human MSLN (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
The immunophenotypes of T cells were tested using flow cytometry. The antibodies used for analysis include: APC-Cy 7 mouse anti-human CD3 (BD, NJ, USA), APC mouse anti-human CD4 (BD, NJ, USA), phycoerythrin (PE) mouse anti-human CD8 (BD, Franklin Lakes, NJ, USA), Brilliant Violet 421 anti-human CD197 (BioLegend, CA, USA), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, San Diego, CA, US), PE-Cy7 anti-human CD45RO (BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD197 (CCR7; BioLegend, San Diego, CA, USA), Brilliant Violet 510 anti-human CD62L (BioLegend, San Diego, CA, USA), Brilliant Violet 605 anti-human CD95 (Fas; BioLegend, San Diego, CA, USA), PE anti-human CD223 (LAG-3; BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD366 (Tim-3; BioLegend, San Diego, CA, USA), and APC anti-human CD279 (PD-1; BioLegend, San Diego, CA, USA). Data were analyzed using NovaExpr software (ACEA, Ashland, OR, USA).
4
Immunophenotyping of Transplant Recipients
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PBMCs were isolated from transplant recipients immediately prior to transplantation (POD 0) and immediately prior to every AT-1501 infusion outlined above. PBMCs were stained with the following antibodies: APC-Cy7 Mouse Anti-Human CD3 (BD Biosciences, clone SP34-2), PerCP-Cy5.5 Mouse Anti-Human CD4 (BD Biosciences, clone L200), PE Mouse Anti-Human CD2 (BD Biosciences, clone RPA-2.10), PE-Cy7 Mouse Anti-Human CD28 (Thermo Fisher Scientific, clone RPA-28.2), BV421 Mouse Anti-Human CD95 (BD Biosciences, clone DX2), APC-Cy7 Mouse Anti-Human CD20 (BD Biosciences, clone L27), PE Mouse Anti-Human CD56 (Thermo Fisher Scientific, clone TULY56), PE-Cy7 Mouse Anti-NHP CD45 (BD Biosciences, clone D058-1283), Alexa Fluor ® 488 anti-human FOXP3 (Biolegend, clone 206D), PE anti-human CD25, Anti-Human (Miltenyi Biotec, clone 4E3), PE-Cy7 CD127 (Thermo Fisher Scientific, clone eBioRDR5), FITC mouse anti-Ki-67 (BD Biosciences). T cell memory compartments were defined for analysis based on CD28 and CD95 expression following identification of CD3 + CD4 + or CD3 + CD8 + T cells. CD28 and CD95 gating to identify naïve (CD28 + CD95 -), central memory (CD28 + CD95 + ) and effector memory (CD28 -CD95 + ) was employed as previously described(68). Samples were acquired with an LSRFortessa flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (version 10).
5
Quantification of Plasma Antibody Responses
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To assess compliance, plasma ARs were analyzed by extraction with diethyl ether and normal phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) after extraction with diethyl ester [30] (link) modified to a 100 mm x 2 mm column and a run time of 8 min. Matrix matched calibration was used, and two control samples were run in triplicate with each batch. Control samples and calibration curve standards were run in random order throughout each sequence. Intra-batch repeatability for each control plasma sample was < 10 % and inter-batch repeatability < 15%. Naïve and memory T lymphocyte subsets. Whole blood in EDTA tubes was stained with a mixture of APC-Cy7 mouse anti-human CD3, FITC mouse anti-human CD4, PerCP-Cy5.5 mouse anti-human CD8, PE-Cy7 mouse anti-human CD45RA and PE rat anti-human CD197 (CCR7) (BD Biosciences, Oxford, UK) in the dark for 45 min at RT. Erythrocytes were lysed using BD Pharm Lyse™ lysing buffer (BD Biosciences, Oxford, UK), incubating in the dark for 20 min at RT, before washing twice with BD CellWASH™ (BD Biosciences). Samples were analysed on a BD FACSCanto II flow cytometer. Results are reported as percentages of parent CD8 + and CD4 + T lymphocyte subsets.
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