Peripheral CD4+ T cells were enriched with the EasySep Human CD4+T cells kit (STEMCELL Technologies) from the blood of patients, heterozygous relatives, and control donors. Enriched CD4+ T cells were stained with the following antibodies: APC-Cy7-anti-CD4, PE-anti-CD25, PerCP-Cy5.5-anti-CD127, AlexaFluor700-anti-HLA-DR (all from BioLegend) and with AlexaFluor647-anti-Helios (BioLegend), Alexa Fluor 488-anti-FOXP3, and eV605-anti-CD3 (both from eBioscience) after fixation and permeabilization of T cells (gating strategy is shown in figs. S23 and S24 ). After staining enriched CD4+ T cells with APC-Cy7-anti-CD4, PE-anti-CD25, and Alexa Fluor 647-anti-CD127 (BioLegend), CD4+CD127+Tconv cells and CD4+CD25hiCD127−/lo Treg cells from patients, heterozygous relatives, and HDs were sorted using a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA). CD4+CD25+CD127+ Tconv cells were labeled with CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) (InVivogen). Cocultures of Treg and Tconv at 1:1, 1:4, 1:8, and 1:16 ratios were stimulated with the Treg Suppression Inspector Human kit (Miltenyi Biotec) at a 1 bead/1 cell ratio. Proliferation of the viable Tconv was analyzed by CFSE dilution at day 3.5.
Percp cy5.5 anti cd127
Manufactured by BioLegend
Sourced in United States
Percp-cy5.5 anti-CD127 is a fluorescent-labeled antibody used for detecting CD127 (also known as IL-7Rα) expression on cells. CD127 is a cell surface receptor that plays a crucial role in lymphocyte development and homeostasis. This antibody can be utilized in flow cytometry applications to identify and quantify CD127-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 apc h7, by BioLegend (2 mentions)
Fitc anti lin, by BD (2 mentions)
Pe cy7 anti crth2, by BioLegend (2 mentions)
Anti cd4 apc cy7, by BioLegend (1 mentions)
Anti cd25 pe, by BioLegend (1 mentions)
Treg suppression inspector human kit, by Miltenyi Biotec (1 mentions)
Alexafluor700 anti hla dr, by BioLegend (1 mentions)
Facsaria, by BD (1 mentions)
Anti cd117 pe, by BioLegend (1 mentions)
Facsaria system, by BD (1 mentions)
Tcrγδ, by BD (1 mentions)
Tcrαβ, by BD (1 mentions)
Tcrαβ, by BioLegend (1 mentions)
Tcrγ δ, by BioLegend (1 mentions)
3 protocols using percp cy5.5 anti cd127
1
Suppressive Function of Regulatory T Cells
2
Isolation and Identification of ILC1s
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ILC1s were sorted from PBMCs using a BD FACSAria. ILC1s were identified as Lin cocktail−(CD3, CD14, CD19, and CD20), CD34−, CD94−, TCRα/β−, TCRγ/δ−, CD1a−, CD45+, CD127+, CRTH2−, and CD117− cells17 ,18 (link). FITC anti-Lin (BD Bioscience, USA), FITC anti-CD94, CD34, CD1a, TCRα/β, TCRγ/δ, APC-H7 anti-CD45, Percp-cy5.5 anti-CD127, PE-Cy7 anti-CRTH2, PE anti-CD117 (Biolegend, USA). Purity was routinely >99%. Cell viability, as determined by trypan blue staining, was >99% after isolation.
3
Isolation and Sorting of Innate Lymphoid Cells
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For further purification, MNCs from freshly resected patient tissue specimens were subjected to Ficoll‐Hypaque gradient centrifugation for 30 min at 24°C. Next, the MNC layer was transferred to a new tube, washed twice with phosphate-buffered saline (PBS), and suspended in PBS. ILCs were sorted using a BD FACSAria system (BD Bioscience) as Lin−-enriched MNCs as Lin cocktail− (CD3, CD19, CD20, and CD14), CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+ CRTH2+/− CD117+/− cells using FITC anti-Lin (643510; BD Bioscience), CD94 (305504; Biolegend, San Diego, CA, USA), CD34 (343504; Biolegend), CD1a (300104; Biolegend), T cell receptor (TCR)α/β (306706; Biolegend), TCRγ/δ (331208; Biolegend), allophycocyanin (APC)-H7 anti-CD45 (56017; Biolegend), Percp-cy5.5 anti-CD127 (351322; Biolegend), phycoerythrin (PE)-Cy7 anti-CRTH2 (350118; Biolegend), and BV605 anti-CD117 (562687; Biolegend). pDCs were sorted as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ BDCA2+ cells using FITC anti-Lin, CD94, CD34, CD1a, TCRα/β, TCRγ/δ, APC-H7 anti-CD45, and APC anti-BDCA2 (17-9818-42; Biolegend). Purity was routinely >99%. Cell viability was determined by trypan blue staining and was >99% after isolation.
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