Cells for flow cytometry analysis were harvested and stained with fluorescently labeled antibodies for 30 min at 4℃ in dark. Following antibodies were used: anti-CD80 (L307.4; BD Biosciences, San Diego, CA), anti-CD32 (FLI8.26; BD Biosciences), anti CD83 (HB15e; Biolegend, San Diego, CA), anti-CD137L (5F4; Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD8 (RPA-T8, Biolegend), anti-CD45RO (UCHL1, Biolegend), anti-CCR7 (G043H7, Biolegend), and anti-CD62L (DREG-26, BD Bioscience). pp65 Tetramer (Proimmune, Oxford, MC, UK) was used for tetramer staining and cells were analyzed with Canto (BD Biosciences). For analysis, lymphocyte population was gated based on FSC and SSC and further gating of CD8+ T cells were done by gating CD3 and CD8 double positive cells. Gating of memory differentiation was done either with CD45RO and CD62L or with CD45RO and CCR7.
Anti cd45ro uchl1
Manufactured by BioLegend
Sourced in Canada
Anti-CD45RO (UCHL1) is a mouse monoclonal antibody that binds to the CD45RO isoform of the human CD45 antigen. CD45 is a transmembrane tyrosine phosphatase expressed on the surface of all nucleated hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 okt3, by BioLegend (1 mentions)
Anti ccr7 g043h7, by BioLegend (1 mentions)
Anti cd8 rpa t8, by BioLegend (1 mentions)
Anti cd80 l307, by BD (1 mentions)
Anti cd25 bc96, by Thermo Fisher Scientific (1 mentions)
Anti cd4 okt4, by BioLegend (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (1 mentions)
Anti foxp3 pch101, by Thermo Fisher Scientific (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Anti siglecf, by BD (1 mentions)
Anti amphiregulin, by R&D Systems (1 mentions)
Anti il 13 ebio13a, by Thermo Fisher Scientific (1 mentions)
Anti il 4 11b11, by BioLegend (1 mentions)
Anti ccr3, by R&D Systems (1 mentions)
Anti cd161 hp 3g10, by BioLegend (1 mentions)
Trfk5, by BioLegend (1 mentions)
Anti cd62l mel 14, by BioLegend (1 mentions)
3 protocols using anti cd45ro uchl1
1
Multiparametric Flow Cytometry Analysis
2
Profiling T Cell Subsets by Flow Cytometry
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient medium (Histopaque-1077; Sigma-Aldrich) using SepMate tubes (Stem-Cell Technologies). Cells were stained with anti-CD4 (OKT4, BioLegend), anti-CD45RO (UCHL1, BioLegend), anti-CD25 (BC96, eBioscience), anti-CD185/CXCR5 (J252D4, BioLegend), anti-CD279/PD-1 (EH12.2H7, BioLegend), anti-CD278/ICOS (C398.4A, BioLegend), anti-CCR6 (G034E3, BioLegend), anti-CXCR3 (G025H7, BioLegend) and anti-Foxp3 (PCH101, eBioscience). Intracellular Foxp3 staining was performed using the Foxp3 Fix/Perm Kit (eBioscience) according to the manufacturer’s instructions. Samples were acquired on a BD LSRFortessa cytometer (BD Biosciences) and further analyzed on FlowJo software (TreeStar).
3
Identification and Analysis of Memory Th2 Cells
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Mouse memory Th2 cells were stained with anti-IL1rL1(ST2) (DIH9, BioLegend), and two subpopulations (ST2 low , ST2 high ) were purified by FACS. Memory Th2 cells were stimulated with immobilized anti-TCRb for 4 hours. Anti-Amphiregulin (206220, R&D Systems) was conjugated by ourselves using an Antibody Labeling Kit (Thermo Fisher Scientific) and used for intracellular staining of mouse samples. Anti-IL-5 (TRFK5, BioLegend), Anti-IL-4 (11B11, BioLegend) and Anti-IL-13 (eBio13A, eBioscience) were used for intracellular staining of mouse samples. Anti-Siglec-F (E50-2440, BD Biosciences), Anti-CCR3 (83101, R&D Systems), Anti-CD101 (Moushi101, eBioscience) and Anti-CD62L (MEL-14, BioLegend) were used for cell surface staining of mouse samples.
T cells from PBMC and dissociated cells from the ECRS polyps were stained with anti-CD4 (OKT4, BioLegend), anti-CD45RO (UCHL1, BioLegend), anti-CD161 (HP-3G10, BioLegend), anti-CRTH2 (BM16, BioLegend), anti-Lineage Cocktails (BioLegend) and anti-CD127 (A019D5, BioLegend). Anti-Amphiregulin (31221, R&D Systems) was conjugated by ourselves using an Antibody Labeling Kit (Thermo Fisher Scientific) and used for intracellular staining for human samples. Anti-IL-5 (TRFK5, BioLegend) and anti-IFNg (4S.B3, BioLegend) were used for intracellular staining of human samples.
T cells from PBMC and dissociated cells from the ECRS polyps were stained with anti-CD4 (OKT4, BioLegend), anti-CD45RO (UCHL1, BioLegend), anti-CD161 (HP-3G10, BioLegend), anti-CRTH2 (BM16, BioLegend), anti-Lineage Cocktails (BioLegend) and anti-CD127 (A019D5, BioLegend). Anti-Amphiregulin (31221, R&D Systems) was conjugated by ourselves using an Antibody Labeling Kit (Thermo Fisher Scientific) and used for intracellular staining for human samples. Anti-IL-5 (TRFK5, BioLegend) and anti-IFNg (4S.B3, BioLegend) were used for intracellular staining of human samples.
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