CD14+ monocytes and CD4+ T cells were isolated from PBMCs by positive selection with antibody-coated microbeads (Miltenyi Biotec). CD14+ monocytes were immediately cryopreserved and stored in liquid nitrogen until required for use as antigen-presenting cells in subsequent stimulation assays. CD4+ T cell subsets were cell sorted to 99% purity on a FACSAria (BD) after staining with FITC-labeled anti-CD45RA (ALB11; Beckman Coulter), allophycocyanin-labeled anti-CD4 (SK3; BD), and anti-CCR7 (150503; R&D Systems), followed by staining with biotinylated anti-IgG2a (SouthernBiotech) and streptavidin–Pacific blue (Invitrogen). PE-cyanine 5 (PC5)–labeled anti-CD56 (N901 [NHK-1]; Beckman Coulter), anti-CD25 (B1.49.9; Beckman Coulter), and anti-CD8 (B9.11; Beckman Coulter) were included as a dump channel to exclude natural killer, regulatory, and CD8+ T cells.
Anti cd25 b1.49.9
Manufactured by Beckman Coulter
Sourced in United States
The Anti-CD25 (B1.49.9) is a monoclonal antibody product from Beckman Coulter. It recognizes the CD25 antigen, which is the alpha subunit of the interleukin-2 receptor expressed on activated T cells and regulatory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin pacific blue, by Thermo Fisher Scientific (1 mentions)
Anti ccr7 150503, by R&D Systems (1 mentions)
Anti cd8 b9.11, by Beckman Coulter (1 mentions)
Antibody coated microbeads, by Miltenyi Biotec (1 mentions)
Facsaria, by BD (1 mentions)
Anti cd4 okt4, by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Anti foxp3 pch101, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque lymphoprep, by Axis-Shield (1 mentions)
Facscanto 2, by BD (1 mentions)
2 protocols using anti cd25 b1.49.9
1
Isolation of CD14+ Monocytes and CD4+ T Cell Subsets
2
Isolation and Analysis of PBMCs
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Peripheral blood mononuclear cells were isolated using Ficoll-Hypaque (Lymphoprep; Axis-shield, Oslo, Norway) and suspended in phosphate-buffered saline containing 0.5% bovine serum albumin (Wako, Osaka, Japan). Anti-CD4 (OKT4; BioLegend, San Diego, CA, USA), anti-CD25 (B1.49.9; Beckman Coulter, Brea, CA, USA), anti-CD45RA (HI100; ALPCO Diagnostics, Salem, NH, USA) and/or anti-FOXP3 (PCH101; eBioscience, San Diego, CA, USA) were used to label PB MNCs after treatment with fixation Permeabilization Buffer (eBioscience). Non-specific control antibodies of the same subclasses were used as negative controls. These PB MNCs were counted by flow cytometry using a FACSCanto II (BD Biosciences), and the data were analyzed with Flow Jo software (FLOW JO LLC, Ashland, OR, USA). For chimerism analysis of CD3+ cells from the thymectomized patient, cells were sorted using EazySep T cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol. The purity of CD3+ cells, evaluated by flow cytometry, was over 99% in each sample.
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