Tregs were purified by magnetic cell sorting (MACS, CD4+CD25+ Regulatory T Cell Isolation Kit, Miltenyi) from peripheral blood mononuclear cells (PBMCs) and cultivated in 96‐well flat plates (1 × 105 cells/well) in complete medium composed of RPMI 1640 and 10% fetal bovine serum, which was supplemented with IL‐2 (500 U/mL, PeproTech), TGF‐β (5 ng/mL, Thermo Fisher) and β‐mercaptoethanol (1:1000, Sigma) in all cultures. Tregs were activated with anti‐CD3/CD28 beads (1:1, Thermo Fisher) for 72 hours. Tregs were stimulated either with TNF‐α (20 ng/mL, Novoprotein) or with TNF‐α and the TNFR2 neutralizing antibody Mab726 (1.5 μg/mL, R&D Systems) for 72 hours. The phenotypes of Tregs were detected by flow cytometry using the following antibodies: anti‐CD4 (FITC, BD), anti‐CD25 (APC, BD), anti‐Foxp3 (PE, BD), anti‐TNFR2 (PE‐CF594, BD), anti‐CTLA‐4 (PerCP‐Cy5.5, Biolegend), anti‐TIGIT (PerCP‐Cy7, Biolegend), anti‐CCR6 (BV510, Biolegend), anti‐ICOS (BV421, Biolegend), anti‐CD44 (PerCP‐Cy5.5, Biolegend), anti‐OX40 (BV510, Biolegend), anti‐41‐BB (BV421, Biolegend), anti‐GITR (PerCP‐Cy7, Biolegend) and anti‐LAP (BV421, Biolegend).
Percp cy7
Manufactured by BioLegend
Sourced in United States
PerCP-Cy7 is a tandem fluorescent dye used in flow cytometry applications. It is composed of the peridinin-chlorophyll protein (PerCP) and the cyanine dye Cy7. PerCP-Cy7 has an excitation maximum at 482 nm and an emission maximum at 778 nm, allowing for its detection in the far-red region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Percp cy5, by BioLegend (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Mab726, by R&D Systems (1 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)
Tnf α, by Novoprotein (1 mentions)
Facscanto 2, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Anti ifn γ clone xmg1, by BioLegend (1 mentions)
Anti granzyme b clone qa16a02, by BioLegend (1 mentions)
2 protocols using percp cy7
1
Regulatory T Cell Activation and Profiling
2
Multiparametric Flow Cytometry Analysis
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Single-cell suspensions were stained with anti-CD45 (clone I3/2,3), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone HMα2), anti-CD73 (clone TY/11.8), anti-PD-1 (clone 29F.1A12), anti-Nrp-1 (clone 3E12), anti-IL-10 (clone JES5-16e3), anti-Foxp3 (clone FJK-165), anti-granzyme B (clone QA16A02), and anti-IFN-γ clone XMG1.2) coupled to FITC, PE, PE-Cy7, PerCP, PerCP-Cy7, and APC (all from Biolegend, NJ, USA). For intracellular cytokine staining, cells were stimulated for 5 h at 37°C adding PMA (50 ng/ml) (Sigma, Milano, Italy), ionomycin (1 mg/ml) (Sigma, Milano, Italy), and Brefeldin A (10 mg/ml) (eBioscience, CA, USA). Then, cells were fixed using a permeabilization kit (Biolegend or BD, USA) according to the manufacturer’s procedure and subsequently stained for intracellular markers. Flow cytometric data were acquired using FACS CantoII (BD Immunocytometry System, San Jose, USA) and analyzed with FlowJo software (Tree Star, OH, USA).
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