Cells were resuspended in PBS supplemented with 2% FCS and stained with a subset of the following mAbs: Pacific Blue anti-CD4; FITC anti-CD25 (BD Biosciences); allophycocyanin anti-FR4; Pacific Blue anti-CD11c; FITC anti-IAb; PE anti-CD40; PerCP anti-CD80; and allophycocyanin anti-CD86 (Biolegend). Cells were acquired using an LSR II flow cytometer (BD Biosciences) at Beth Israel Deaconess Medical Center, Flow Cytometry Core, and the results were analyzed with FlowJo 8.7 software (Treestar). To determine the number of Treg cells (CD4+CD25+Foxp3+FR4+) in vivo, 1 × 106 cells were stained for the intracellular transcription factor Foxp3 using allophycocyanin anti-mouse/rat Foxp3 (eBioscience). For intracellular cytokine staining, 1 × 106 cells were stimulated in vitro for 4 h at 37°C in 5% CO2 with phorbol-12-myristate-13-acetate (PMA, 100 ng/mL), ionomycin (1 μg/mL), and brefeldin-A (1 μg/mL) (Sigma-Aldrich). The cells were permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization solution kit (BD Biosciences). Intracellular staining was performed with allophycocyanin anti-IL-17 and FITC anti-IFN-γ (Biolegend).
Anti cd25 fitc
Manufactured by BD
Sourced in United States
Anti-CD25-FITC is a fluorescent-labeled antibody that binds to the CD25 antigen, also known as the interleukin-2 (IL-2) receptor alpha chain. It can be used in flow cytometry applications to identify and quantify cells expressing the CD25 marker.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 pe, by BD (9 mentions)
Anti cd4 apc, by BD (8 mentions)
Anti cd4 percp, by BD (4 mentions)
Anti cd127 pe, by BD (4 mentions)
Anti cd3 percp, by BD (4 mentions)
Facscalibur, by BD (3 mentions)
Anti cd8 fitc, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Anti cd14 fitc, by BD (3 mentions)
Anti cd3 pe, by BD (3 mentions)
Anti cd19 apc, by BD (3 mentions)
Anti cd8 pe cy5, by BD (3 mentions)
Pe cy5 anti cd4, by BD (3 mentions)
Facsaria, by BD (2 mentions)
Anti cd45ra fitc, by BD (2 mentions)
Anti cd45ro pe, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Anti cd69 fitc, by BD (2 mentions)
Anti cd62l apc, by BD (2 mentions)
29 protocols using anti cd25 fitc
1
Comprehensive Immune Cell Phenotyping
2
Investigating Ezh2 Deficiency in T Cell-Mediated Colitis
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CD4+ T cells were enriched from the spleen and lymph nodes of control (Ezh2fl/fl) and Ezh−/− (Cd4 Cre; Ezh2fl/fl) mice with an AutoMACS cell separator (Miltenyi Biotec), stained with PerCP Cy5.5 anti-CD4, FITC anti-CD25, and PE anti-CD45RB (all obtained from BD Biosciences), and naive CD4+ CD25− CD45RBhi T cells were purified (>99%) by cell sorting (Moflo,Dako Cytomation or FACSAria, BD Biosciences). CD4+GFP+CD25+NRP1+ tTreg cells were purified from Ezh2fl/flFoxp3-GFP mice or Cd4 Cre; Ezh2fl/flFoxp3-GFP. CD4+CD45RBhiCD25− naive T cells (4 × 105) from control mice and Ezh2-deficient mice were injected intravenously (i.v.) into age- and sex-matched Rag2−/− mice and intestinal inflammation was monitored. Alternatively, Rag2−/− mice were coinjected with 4 × 105 naive T cells and 1 × 105 CD4+CD45RBloCD25+ Treg cells from control mice and Ezh2-deficient mice. Mice were sacrificed at 7 weeks when significant weight loss occurred in the control groups. Gut cells were isolated as described previously by intracellular staining. Sections of the proximal, mid-, and distal colon were fixed in buffered 10% formalin and stained with hematoxylin and eosin (H&E) (Histoserv).
3
CD4+CD25+ T Cell Activation Assay
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Isolated CD4+CD25+ T cells were incubated for 30’ at 37°C in 5% CO2 with or without 10 μM of Peptide R29. After 30’ the cells were washed and then stained with Fitc-anti-CD25, APC-Cy7-anti-CD4 (BD Bioscience) and 7-amino-actinomycin D (7-AAD) (BioLegend) and analyzed by flow cytometer.
4
Molecular Mechanisms of Inflammation
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The oxazolone and sulfasalazine were purchased Sigma-Aldrich (St. Louis, MA, USA). Primary antibodies for western blot including phosphorylated (p)–NF–κB (Catalog No. 3033), and NLRP3 (Catalog No. 15101), and horseradish peroxidase-linked secondary antibody (Catalog No. 7074) were purchased Cell Signaling Technology (Danvers, MA, USA). The conjugated fluorescence antibodies for flow cytometry analysis (FACS) including PE-Cy5 anti-CD3, PE anti-CD4, fluorescein isothiocyanate (FITC) anti-CD8, FITC anti-CD19, and FITC anti-CD25, were obtained BD Biosciences (San Diego, CA, USA). All other chemicals and reagents were obtained from Sigma Aldrich.
5
Phenotypic Analysis of Regulatory T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood and BM mononuclear cells (BMMCs) were obtained from BM in both of OA and RA patients by Ficoll-Paque PLUS (GE Healthcare Biosciences AB, Uppsala, Sweden) density centrifugation. To examine the phenotype of regulatory T cells (Tregs), the following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD45RA-FITC, anti-CD45RO-PE and anti-CD184 (CXCR4)-PE (all from BD Biosciences, San Jose, CA, USA). Next, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set followed by intracellular staining with anti-Foxp3-APC antibody (PCH101; eBioscience, San Diego, CA, USA). Cells were acquired using FACSCalibur (BD Biosciences), and the results were analyzed using CellQuest (BD Biosciences) software. The gating strategy was based on the identification of lymphocytes according to FSC and SSC signal distribution. Then, CD4+ lymphocytes were gated, and next, appropriate cell populations were identified. Gates were settled according to the isotype control or fluorescence minus one (FMO) for the desired marker. The dot plots shown are representative for at least six experiments done on different patients. The described gating strategy is shown in Figure 2b and was used though all the analysis of the cell phenotype presented in this paper.
6
NK Cell Activation Analysis
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NK primary cells, treated with 4c compound or with DMSO vehicle control for 24 hours, were labeled with anti-CD25 FITC, anti CD38-APC (BD) and analyzed by FACSCalibur cytometer (BD).
7
Multiparametric PBMC Phenotyping
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PBMCs were isolated by Histopaque-1077 (Sigma Aldrich, USA) density gradient centrifugation and, after gating for CD3 positivity, were stained with anti-CD4 PE, anti-CD25FITC (BD Biosciences, USA), anti-CD69 and anti-FoxP3 for FACS fluorescence cytometry. After gating for Lin-negativity, the isolated PBMCs were also stained for the myeloid dendritic markers CD11c and CD86PE (eBioscience, USA). The data were analyzed with a Beckman Coulter flow cytometer.
8
Comprehensive Immune Cell Profiling
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Ab-stained cells were analyzed using FACScaliber (BD Biosciences, USA) CellQuest Software. Anti-CD4-PE, anti-CD4-APC, anti-CD8-FITC, anti-CD25-FITC, anti-CD44-PE, anti-CD62L-APC, anti-CD69-FITC, anti-CCR5-PE, anti-CXCR3-PE, and anti-CXCR4-PE, anti-IFNγ-BV421, anti-IL17A-PE, anti-Thy1.2-BV605 were purchased from BD Biosciences.
9
Immune Modulatory Nanoparticle Synthesis
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Fibrinogen, FITC-BSA conjugate, uricase (Candida sp.), and all chemicals were purchased from Sigma-Aldrich unless otherwise noted and were used as received. Pierce 660 nm Protein Assay Reagent was purchased from Thermo Fisher Scientific. Goat anti-mouse IgM antibody and goat anti-mouse IgG antibody were purchased from Bethyl Laboratories Inc. TNF-α, IL-4, IL-6, IL-10, CXCL-10, and TGF-β Quantikine ELISA kits were purchased from R&D Systems. Anti–CD40-FITC, anti–CD80–phycoerythrin (PE), anti–CD4-PE, anti–CD25-FITC, and anti–Foxp3-Percep antibodies were purchased from BD Bioscience. PEG polymers with different molecule weights were purchased from BroadPharm. Mouse macrophages RAW 264.7 were purchased from American Type Culture Collection. Mouse dendritic cell line DC 2.4 was received from K. L. Rock (University of Massachusetts Medical Center, Worcester, MA) as a gift.
10
Osteoblast Purity and Immune Cell Profiling
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Osteoblasts were acquired and washed with PBS three times. All samples were divided into control and test tubes. Antibodies against mouse IgG1-FITC, IgG1-PE and IgG1-PerCP (BD Biosciences, US) were stained as a negative control. Antibodies against CD45-PerCP, CD138-FITC, and CD34-PE (BD Biosciences, US) were stained to identify the purity of osteoblasts. Peripheral blood samples were collected in EDTA- anticoagulant tubes. The number of immune subsets were measured by FCM using anti-CD4-FITC, anti-CD8-FE and anti-CD3-PerCP (to identify the subtypes of T lymphocytes); anti-CD4-PE, anti-CD25-FITC and anti-CD127-APC (regulatory T cells); anti-Lin-FITC, anti-HLA-DR-PerCP, anti-CD11c-PE and anti-CD123-APC (subtypes of dendritic cells, DC); anti-CD3-APC, anti-CD8-PerCP, anti-IFN-γ-FITC and anti-IL-4-PE (T helper cells, Th) (BD Biosciences, US). After incubation in the dark at 4°C for 30 min, the cells were incubated with 2 ml erythrocyte lytic solution (BD Biosciences) at room temperature for 10 min. The cells were then washed two times with PBS. At least 10,000-30,000 cells were acquired and analyzed on FACSCalibur flow cytometer (BD Biosciences).
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