Cultured MNCs were collected from each well and centrifuged. The cell pellet was resuspended in PBS. The sample to be tested was incubated with the following antibodies at 4 °C for 30 min. CD45+ lymphocytes, CD3+ T cells, CD3+ CD4+ T cells, and CD3+ CD8+ T cells were detected with the FITC anti-CD3/PE anti-CD8/PerCP anti-CD45/APC anti-CD4 detection kit (ACEA Biosciences, China). CD3− CD56+ CD16+ NK cells and CD3− CD19+ B cells were detected using a FITC anti-CD3/PE anti-CD16+ CD56/PerCP anti-CD45/APC anti-CD19 detection kit (ACEA biosciences). CD4+ CD25+ Foxp3+ Treg cells were detected with FITC anti-human-CD25 (BioLegend), PerCP/Cy5.5 anti-human-CD4 (BioLegend), and PE anti-human-Foxp3 (BioLegend). CD38+ CD3− CD56+ NK cells were detected with PE anti-CD38 (BioLegend), FITC anti-CD3 (BioLegend), and APC anti-CD56 (BioLegend). Lymphocyte subtypes within MNCs were measured with flow cytometry.
Percp cy5.5 anti human cd4
Manufactured by BioLegend
PerCP/Cy5.5 anti-human-CD4 is a fluorochrome-conjugated antibody that binds to the CD4 surface antigen expressed on human T helper cells. It can be used for the identification and enumeration of CD4+ T cells in flow cytometric applications.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti human cd25, by BioLegend (3 mentions)
Fe anti human cd127, by BioLegend (2 mentions)
Facsdiva v6, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
Anti cd38 pe, by BioLegend (1 mentions)
Pe anti human foxp3, by BioLegend (1 mentions)
Anti cd56 apc, by BioLegend (1 mentions)
Perm wash, by BD (1 mentions)
Anti human il 17a apc, by Thermo Fisher Scientific (1 mentions)
Fitc mouse anti human ifn γ, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti human cd3 fitc, by BioLegend (1 mentions)
Human regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Fcr block, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Saponin, by Merck Group (1 mentions)
Cellquest pro software, by BD (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
5 protocols using percp cy5.5 anti human cd4
1
Multiparametric Flow Cytometry Analysis
2
Isolation and Characterization of Regulatory T Cells from RA Patients
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MNCs were obtained from the peripheral blood of RA patients (n = 9, from patient no. 13 to no. 21). The MNCs were incubated with FITC anti-human-CD25 (BioLegend), PerCP/Cy5.5 anti-human-CD4 (BioLegend), and FE anti-human-CD127 (BioLegend) antibodies for 30 min at room temperature. The cells were collected by centrifugation and resuspended in PBS. The labeled cells were sorted using a BD FACS ARIA II with BD FACS Diva V6 software (BD Biosciences, USA). The Treg cells were analyzed for purity. This method is common for Treg cell preparation [35 ].
3
Cytokine Profiling of Activated T Cells
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After three days of stimulation the CD3/CD28 beads were removed from the cells, and the cells were re-stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of monensin (3 µM) as previously described [59] . The cells were then stained with PerCP/Cy5.5 anti-human CD4 (317428, BioLegend), fixed and permeabilized with BD Cytofix/Cytoperm followed by BD Perm/Wash according to the manufacturer instructions, and finally stained intracellularly with FITC mouse anti-human IFN-γ (554551, BD Pharmingen), anti-human IL-17A APC (17–7179, eBioscience), FITC Rat Anti-Human IL-4 (554484, BD Pharmingen) or PE anti-human IL-13 (501903, BioLegend). Data were acquired on a FACSCalibur (BD, Brøndby, Denmark) with CellQuest Pro software, and subsequently analyzed using FlowJo software.
4
Isolation and Flow Cytometry Analysis of IL-17+ Cells
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Peripheral blood mononuclear cells of heparinized peripheral blood from the study subjects were isolated by Ficoll density gradient centrifugation (HAO YANG, Tianjin, China). Flow cytometry analysis of IL‐17‐producing cells was carried out as previously described.18 Briefly, 2 × 106 PBMC were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin in the presence of 10 μg/mL brefeldin A (BioLegend, San Diego, CA, USA) in 24‐well plates for 4 hours. Cells were harvested, washed, and stained with FITC‐antihuman CD3, or PerCP/Cy5.5‐antihuman CD4, or PerCP/Cy5.5‐antihuman CD8, or PerCP/Cy5.5‐antihuman TCR‐γδ (all from BioLegend) for 30 minutes on ice in the presence of FcR‐block (BioLegend). After washing, cells were fixed with 4% paraformaldehyde and permeabilized with 1% saponin (Sigma Chemical Co., St Louis, MO, USA), and then stained with phycoerythrin (PE)‐antihuman IL‐17 and Allophycocyanin (APC)‐antihuman IFN‐γ (all from BioLegend) for 30 minutes on ice. Treg cell staining was carried out with a Human Regulatory T Cell Staining Kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. Treg cells were defined as CD3+CD4+Foxp3+ cells. Data were acquired on a FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using CellQuest Pro software (BD Biosciences).
5
Measuring Treg Cell Suppressive Function
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MNCs were obtained from the peripheral blood of RA patients. The MNCs were incubated with FITC antihuman-CD25 (BioLegend), PerCP/Cy5.5 anti-human-CD4 (BioLegend), and FE anti-human-CD127 (BioLegend) antibodies for 30 min at room temperature. The cells were collected by centrifugation and collected in PBS. The labeled cells were sorted using a BD FACS ARIA II with BD FACS Diva V6 software (BD Biosciences, USA). The Treg cells were analyzed for purity.
Cell count kit-8 (CCK8) assay for Treg cells CD38 + NK or CD38 + NKT cells were treated with CD38 monoclonal antibody (CD38 mAb, TheraMabs Bio-Technology, China) at a nal concentration of 1 µg/mL or an equal volume of PBS at 37 °C for 24 h. CD38 + NK or CD38 + NKT cells were then washed and cocultured with Treg cells in a transwell apparatus at 37 °C and 5% CO 2 for 24 h. Treg cells were suspended and washed, and CCK-8 solution (Dojindo, Japan) was added to the 90 µl culture. The OD values were measured at 450 nm using a spectrophotometer (BioTek, USA) after incubation for 3 h.
Cell count kit-8 (CCK8) assay for Treg cells CD38 + NK or CD38 + NKT cells were treated with CD38 monoclonal antibody (CD38 mAb, TheraMabs Bio-Technology, China) at a nal concentration of 1 µg/mL or an equal volume of PBS at 37 °C for 24 h. CD38 + NK or CD38 + NKT cells were then washed and cocultured with Treg cells in a transwell apparatus at 37 °C and 5% CO 2 for 24 h. Treg cells were suspended and washed, and CCK-8 solution (Dojindo, Japan) was added to the 90 µl culture. The OD values were measured at 450 nm using a spectrophotometer (BioTek, USA) after incubation for 3 h.
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