Peripheral blood mononuclear cells (PBMCs) in SLE patients and healthy donors were analyzed by flow cytometry (FACSAria II; BD Biosciences). To analyze IL-2 production, PBMCs were stimulated and incubated for 4 h with 50 ng/mL phorbol myristate acetate, 1 g/mL ionomycin (both from Sigma-Aldrich), and 1 L/mL GolgiStop (BD Biosciences). After stimulation, the cell surface markers CD4, CD3, CD8, CD56, CD25 and CD127 were stained with the fluorescence labeled monoclonal antibodies. Next, the cells were fixed and permeabilised for 30 min at 4°C at dark with BD FACS fixation and permeabilisation buffer set (BD Biosciences). Then the cells were stained with PE labeled anti-human-IL-2 antibody. Proportion of IL-2 expressing cells in CD4+ effector T cells (defined as CD4+ CD8− CD127hi/low CD25low/−), double-negative T cells (DNT cell, CD3+ CD4− CD8−), NK cells (CD56+ CD3−) and NKT cells (CD56+ CD3+) was analyzed using FlowJo v10 software (Tree Star). Antibodies used for flow cytometry included APC-H7-anti-human CD3 (Biolegend), Percp-anti-human CD8 (Biolegend), FITC-anti-human CD4 (Biolegend), BV421-anti-human CD25 (Biolegend), BV605-anti-human CD127 (Biolegend), APC-anti-human CD56 (Biolegend) and PE-anti-human IL-2 (Biolegend).
Percp anti human cd8
Manufactured by BioLegend
PerCP anti-human CD8 is a fluorescently labeled antibody that binds to the CD8 surface receptor on human T cells. It is designed for use in flow cytometry applications to identify and quantify CD8-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti human cd4, by BioLegend (2 mentions)
Golgistop, by BD (2 mentions)
Apc anti human cd56, by BioLegend (1 mentions)
Pe anti human il 2, by BioLegend (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
Zombie nir fixable viability kit, by BioLegend (1 mentions)
Alexa fluor 647 anti human ifnγ, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Alexa fluor 488 anti human cd3, by BioLegend (1 mentions)
Apc anti human cd4, by BioLegend (1 mentions)
Pe anti human cd56, by BioLegend (1 mentions)
Apc anti human cd69, by BioLegend (1 mentions)
Human trustain fcx fc receptor blocking solution, by BioLegend (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lectin from phaseolus vulgaris, by Merck Group (1 mentions)
8 protocols using percp anti human cd8
1
IL-2 Production in Immune Cells of SLE Patients
2
Cryopreserved PBMC Stimulation Assay
3
Flow Cytometric Analysis of CD4+ and CD8+ T Cells
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The sample used was peripheral blood mononuclear cell (PBMC), which was isolated from peripheral blood. Antibodies used are Biolegend FITC anti-human CD4 (300506) and Biolegend PerCp anti-human CD8 (344708). Further, 10
3to 10
5PBMC was added to specific primary antibodies with optimum concentration. The sample was incubated for 15 to 20 minutes in a dark room. After washing twice, to the sample was added secondary antibodies, namely fluorochrome conjugated anti-human immunoglobulin (FITC anti-human), and PerCP incubation was done on ice in a dark room for 15 to 20 minutes, then washed again. The pellet cells were resuspended in 0.5 mL cell-staining buffer and were added 5 μL (0.25 μg) viability-staining solution to remove the dead cells. Measurements were made at 10
5PBMC, and the results obtained in the form of a percentage (%) of cells were analyzed using BD Cell Quest Pro software.
19
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The sample used was peripheral blood mononuclear cell (PBMC), which was isolated from peripheral blood. Antibodies used are Biolegend FITC anti-human CD4 (300506) and Biolegend PerCp anti-human CD8 (344708). Further, 10
3to 10
5PBMC was added to specific primary antibodies with optimum concentration. The sample was incubated for 15 to 20 minutes in a dark room. After washing twice, to the sample was added secondary antibodies, namely fluorochrome conjugated anti-human immunoglobulin (FITC anti-human), and PerCP incubation was done on ice in a dark room for 15 to 20 minutes, then washed again. The pellet cells were resuspended in 0.5 mL cell-staining buffer and were added 5 μL (0.25 μg) viability-staining solution to remove the dead cells. Measurements were made at 10
5PBMC, and the results obtained in the form of a percentage (%) of cells were analyzed using BD Cell Quest Pro software.
19
4
BiTE-Induced T Cell Proliferation
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PBMCs of two healthy donors were seeded into 96-well U type plate and incubate with DR30318 or AMG910 analog for 0,3 or 7 days. Then, Alexa Fluor® 488 anti-Human CD3 (Biolegend, 317,310), PerCP anti-human CD8 (Biolegend, 344,708) and APC anti-human CD4 (Biolegend, 300,514) were applied for measurement of the BiTE—induced T cell proliferation.
5
Multiparametric Analysis of Human Lymphocytes
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FITC mouse anti-human CD3 (BioLegend; #300306), PerCP anti-human CD8 (BioLegend; #344708), PE anti-human CD56 (BioLegend; #318306), APC anti-human CD69 (BioLegend; #310910), PE/Cy7 anti-human CD11a (BioLegend; #301220), and corresponding IgG isotype controls were used for staining the lymphocytes. The lymphocytes were twice washed with FACS Buffer (1× PBS, 2% heat inactivated FBS) and centrifuged at 1600 RPM for 10 min at room temperature. The washed lymphocytes were blocked for 10 min in FACS Buffer and Human Trustain FcX Fc receptor blocking solution (Biolegend; #422302) at room temperature. The primary antibodies were used as per the manufacturer's instructions and incubated with the cells for 1 h at room temperature. The cells were then washed three times with FACS buffer, and they were then analyzed by flow cytometry at the VCU Flow Cytometry Core.
6
Flow Cytometric Lymphocyte Immunophenotyping
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One hundred microliters of peripheral blood were collected and analyzed using the BD FACS Aria flow cytometer. Antibodies used are Biolegend FITC anti-human CD4 and Biolegend PerCp anti-human CD8. In addition, 103–105 PBMC were added to specific primary antibodies at the optimal concentration. The sample was incubated in a dark room for 20 min. After washing twice, the secondary antibody, fluorochrome-conjugated anti-human immunoglobulin anti-human (FITC anti-human), and PerCp were added to cells, followed by incubation on ice in a dark room for 15–20 min and then washing. Data acquired from the flow cytometer were analyzed using BD FACS diva software. The lymphocyte population was gated and then the lymphocyte subsets were gated: CD3 + CD4 + helper T cells and CD3 + CD8 + cytotoxic T cells.
7
Multicolor Flow Cytometry Staining Panel
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Anti-human CD4-FITC, anti-human CD19-FITC, goat F(ab’)2 anti-human-IgM-RPE, and goat F(ab’)2 anti-human-lambda-RPE were from SouthernBiotech. Anti-human CD8-PerCp and anti-human CD56-Alexa-Fluor 700 were purchased from Biolegend. CellTrace Violet Cell Proliferation Kit, LIVE/DEAD Fixable Aqua Dead cell stain kit and anti-human CD3 Brilliant Violet 421 were from Life Technologies, and anti-human FAIM3 (hFCMR, clone 1E4 mouse IgG2bκ) mAb was from Abnova (Taiwan). IgM from human serum, human serum from AB male, and lectin from Phaseolus vulgaris (phytohemagglutinin; PHA) were all from Sigma-Aldrich. Biotinylated Sambucus Nigra Lectin (SNA) was from Vector Laboratories, and biotinylated F(ab’)2 anti-human IgM was from Jackson ImmunoResearch. The structure, function and purification of domain swapped antibodies have been described previously16 (link)17 (link)18 (link).
8
Preparation and Characterization of AFP TCR-T Cells
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The preparation method of AFP TCR-T cells was according to our previous study (22 (link)). The ratio of AFP specific T cells was detected by flow cytometry with anti-mouse TCR vβ-FITC and anti-human CD8-PerCP (BioLegend). Then the Mock or AFP TCR-T cells were co-cultured with HepG2 cells, which were infected with lentiviruses expressing empty vector or LAGE3-shRNA at an effector-to-target cell (E:T) ratio of 2:1. Three hours later, the immunofluorescence stain of HepG2 cells was done with anti-rabbit cleaved-caspase3 (9664S, CST) at 1:400 and AlexaFluor 488-conjugate goat anti-rabbit IgG (ZF0511, ZSGB-BIO, China) at 1:1000. After 12 h of coculture, the cytotoxicity of T cells was detected by measuring the lactate dehydrogenase (LDH) activity according to the manufacturer’s protocol (Promega, USA). And the IFN-γ and TNF-α levels in the culture supernatant was detected using enzyme-linked immunosorbent assay (ELISA) kit (Biolegend).
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