PBMCs were isolated from whole blood cells using Ficoll media purchased from Haoyang Bio-Manufacture CO., LTD (Tianjin, China). Staining fixable viability stain 780 (BD#565388) was added into the cell suspension at 1:1000, followed by an incubation with human Fcblock (BD#564220). Surface markers were stained using CD3-PerCP-Cy5.5 (BD#560835), CD4-PE-Cy7 (BD#560649), CD8-BV510 (BD#563256), CD25-PE (BD#557138), CD127-AF647 (BD#558598), CD45RA-APC (BD#550855) and CCR7-FITC (BD#560548). For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD#554715) and then the cytokines were stained using IL-4-APC (BD#560671), IFN-γ-FITC (BD#554700), TNF-α-BV421 (BD#562783), and IL-17-PE (BD#560487), before the final flow cytometry assay using BD CantoII. Raw data of flow cytometry was analyzed by FlowJo software.
Cd127 af647
Manufactured by BD
Sourced in United States
CD127-AF647 is a fluorescently-labeled antibody that binds to the CD127 (IL-7 receptor alpha chain) surface antigen. It can be used for the identification and analysis of cells expressing CD127 in flow cytometry and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe cy7, by BD (3 mentions)
Cd25 pe, by BD (2 mentions)
Cd3 apc h7, by BD (2 mentions)
Human fc block, by BD (1 mentions)
Il 17 pe, by BD (1 mentions)
Il 4 apc, by BD (1 mentions)
Ifn γ fitc, by BD (1 mentions)
Cd8 bv510, by BD (1 mentions)
Tnf α bv421, by BD (1 mentions)
Ccr7 fitc, by BD (1 mentions)
Cd45ra apc, by BD (1 mentions)
Cd3 percp cy5, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Fixable viability stain 780, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Phytohaemagglutinin pha, by Merck Group (1 mentions)
7 aad, by BD (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
5 protocols using cd127 af647
1
Multiparameter Flow Cytometry of PBMCs
2
PBMC Proliferation Assay with IL-7 and sIL-7RA
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PBMC were incubated with CFSE (1.5 μM) for 10 minutes at 37 °C. PBMC (2 × 106 cells/μL) were cultured for seven days with autologous serum and with either 25 or 50 ng/mL IL-7 (Peprotech), 104 U/mL IL-2 or 5 μg/mL phytohaemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO, USA). Furthermore, in a total of ten participants (five ‘CC’ and five ‘TT’) proliferation was assessed in cells cultured with IL-7 (25 ng/ml) plus sIL-7RA in three different ratios (1 to 100, 1 to 1000 or 1 to 2000) in PBMC). After seven days the cells were incubated with antibodies (CD3-APC-H7, CD4-PeCy7, CD127-AF647, and 7-AAD) (all purchased from BD) and assessed by flow cytometry. The data are given as the proportion of CD4+ T cells and the proportion of CD4 + CD127+ T cells that has made at least one cell division (representative plots in Fig. 3A –E).
3
Tolerogenic Dendritic Cell Modulation of T-Cell Subsets
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After 5 days of culture, PBMC co-cultured with mature DC (mDC), VitD3-tolDC (100% VitD3-tolDC) and 50% VitD3-tolDC (50% VitD3-tolDC + 50% mDC) in the presence and absence of IFN-beta were harvested and stained using CD3-V450, CD4-PerCP-Cy5.5, CD45RA PE-Cy7, CCR7 PE, CD38 APC, CD8 APC-H7, HLA-DR V500 (BD Bioscience), CD183 AF488, CD196 BV605 and CD45 AF700 (Biolegend, San Diego, CA, USA) for T cell analysis; and CD4 PerCP-Cy5.5, CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, CD3 V450, HLA-DR V500 (BD Biosciences) and CD45 AF700 (Biolegend) for Treg analysis. Monoclonal antibodies were incubated for 20 min at room temperature and protected from the light. Samples were washed and a total of 50,000 CD3+ events were acquired on an LSR Fortessa flow cytometer (BD Biosciences). Both panels were analyzed using FACSDiva software (BD Biosciences). The gating strategy used to analyze the desired T cell subpopulations was previously reported [28 (link)].
4
IL-7 Binding to CD4+ T Cells
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Fluorokine biotinylated human IL-7 (NF700, R&D Systems, Abingdon, UK) was used to determine the binding of IL-7 to CD4 + CD127+ T cells according to manufacturer’s instruction. In brief, biotinylated IL-7 and 105 PBMC suspended in 25 μL PBS were incubated at 5 °C for 90 min followed by incubation with avidin-FITC (R&D Systems) and the monoclonal antibodies CD3-APC-H7, CD4-PeCy7, and CD127-AF647 (all purchased from BD), and assessed by flow cytometry. A biotinylated non-reactive protein (soybean trypsin inhibitor, R&D systems) was used as a negative control, and specificity was tested by adding a polyclonal IL-7 blocking antibody (R&D) (representative plots in Fig. 1A –D).
5
Polychromatic Flow Cytometry Analysis
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The following fluorochrome-conjugated Abs were used for polychromatic flow cytometry analysis: CD3-Pacific Blue (UCHT1), CD4-Alexa700 (RPA-T4), CD45RA-APC-Cy7 (HI100), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CD25-PE (M-A251), CD26-FITC (L272), CD127-AF647 (HIL-7R-M21), CD161-PE-Cy5 (DX12), IFNγ-Alexa 700 (B27), CCR5-PE (2D7), CXCR4-PE (12G5), and CD8-APC H7 (SK1) (BD Pharmingen), CD45RA-APC eFluor780 (HI100), CD56-FITC (MEM188), IL-17A-PE (eBio64DEC17), FoxP3-AF488 (PCH101), TNFα-Pacific Blue (Mab11), and IL-17A-eFluor660 (eBio64CAP17) (eBioscience), CD8-FITC (BW135/80), CD19-FITC (LT19) (Miltenyi), CCR7-PE (150503) (R&D) and CD31-BV605 (WM59) (Biolegend). Cell phenotype was analyzed by flow cytometry using the BD LSRII cytometer and BD Diva software. A viability staining Vivid (Invitrogen) was included in each staining cocktail to exclude dead cells from our analysis. FACS analysis was performed using the FlowJo software (©Tree Star, Inc.). For multicolor analysis, all Abs were titrated for an optimal noise/signal ratio and Abs cocktails were validated by comparing single to multiple staining. Positivity gates were placed based on fluorescence minus one (FMO), as previously described [21 (link),101 ].
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