Single cell suspensions of bone marrow (BM) cells and splenocytes were prepared as described previously50 (link). All staining was performed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco Life Technologies) supplemented with 2% fetal bovine serum (FBS; Sigma) on ice. Cells were blocked using FcR blocking reagent (Miltenyi Biotech, 1:20) and stained with combinations of the antibodies listed in Table S27 . Lymphoid progenitors were analysed using APC-mouse lineage antibody cocktail (558074, BD Biosciences), FITC anti-CD127 (IL7-Ra), PE-Cy7 anti-c-Kit and PB anti- Sca1 (Table S27 ). Dead cells were excluded by gating on 7AAD (420403, Biolegend) negative cells. Fluorescence-minus-one controls were used to set appropriate gates. Flow cytometry analysis were performed on a CyAn ADP flow cytometer (Dako) or an LSR Fortessa cell analyser (BD Biosciences) and data were analysed with FlowJo software v 10 (Tree Star).
Anti cd127 fitc
Manufactured by BD
Sourced in United States
Anti-CD127-FITC is a fluorescently labeled monoclonal antibody that binds to the CD127 (IL-7 receptor alpha) antigen. It is used for the identification and enumeration of CD127-positive cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa cell analyser, by BD (2 mentions)
7 aad, by BioLegend (2 mentions)
Apc mouse lineage antibody cocktail, by BD (2 mentions)
Cyan adp flow cytometer, by Agilent Technologies (2 mentions)
Anti cd14 apc cy7, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd34 apc, by BD (1 mentions)
Pecy7 anti ckit, by BD (1 mentions)
Anti sca 1 apc cy7, by BD (1 mentions)
Anti cd48 fitc, by BioLegend (1 mentions)
Anti ps6k thr389, by Cell Signaling Technology (1 mentions)
Anti rabbit igg pe, by Cell Signaling Technology (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd8 pe, by BD (1 mentions)
Anti cd62l apc, by BD (1 mentions)
Anti cd3 bv605, by BioLegend (1 mentions)
7 protocols using anti cd127 fitc
1
Multiparametric Flow Cytometry Analysis
2
Multiparametric Flow Cytometry Analysis
3
Multiplex Flow Cytometry of Hematopoietic Stem Cells
4
Multiparameter Flow Cytometry Analysis
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Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).
5
Profiling HBV-specific CD4+ T Cells
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PBMCs were stimulated with HBV core 18–27 epitope (HBc 18–27, sequence: FLPSDFFPSV; final concentration: 10 μg/mL) in the presence of Brefeldin A (final concentration: 10 μg/mL) for 12 h. Cells were harvested and transferred to FACS tubes, and were stained with anti-CD4-PerCP (BD Bioscience, San Jose, CA, USA), anti-CD25-APC (BD Bioscience), and anti-CD127-FITC (BD Bioscience) for 20 min in the dark at 4 °C. Cells were washed twice with Staining Buffer (BD Bioscience), and were resuspended with 250 μL of Fixation/Permeabilization solution (BD Bioscience) for 20 min at 4 °C. Cells were then washed twice in 1 × BD Perm/Wash buffer (BD Bioscience), and were stained with anti-IL-17-PE (BD Bioscience) for 30 min in the dark at 4 °C. In certain experiments, purified CD4+CD25+CD127dim/− Tregs were stained with anti-CCR4-FITC (BD Bioscience) and anti-CCR6-PE (BD Bioscience) for 20 min in the dark at 4 °C. Isotype antibodies were used to separate positive and negative cells in PerCP, APC, FITC, and PE fluorescence channels. Samples were analyzed with FACS Calibur analyzer (BD Bioscience). Acquisitions were performed with CellQuest Pro Software (BD Bioscience), and analyses were performed with FlowJo Version 8.4.2 for Windows (Tree Star, Ashland, OR, USA).
6
Isolation and Flow Cytometric Analysis of ILC2s
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Lung tissue was digested in 50 μg/mL Liberase TM (1:100) + 1 μg/mL DNase I (1:200). A cell suspension was collected. To detect ILC2s, antibodies for surface antigens were incubated for 30 min, resuspended in FACS buffer and separated on a LSR-Fortessa (BD Pharmingen, San Diego, CA, USA) based on the surface-marker expression. The following antibodies were used: lineage-APC Streptavidin (BD Pharmingen) markers (CD3ε (BD Pharmingen), CD4 (eBioscience, San Diego, CA, USA), CD8a (BD Pharmingen), CD11c (eBioscience), FceRIa (BioLegend), NK1.1 (BioLegend), CD19 (eBioscience), TER119 (eBioscience), CD5 (BD Pharmingen), F4/80 (eBioscience), Ly-6G and Ly-6C (Gr-1, BD Pharmingen)), anti-7AAD (BD Pharmingen), anti-CD45-APCcy7 (BD Pharmingen), anti-ST2-PE (BD Pharmingen) and anti-CD127-FITC (BD Pharmingen).
7
Comprehensive Flow Cytometry Analysis of PBMC
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Flow cytometry analysis was performed prospectively using samples from a part of patients at treatment initiation and 6 months post-treatment. Peripheral blood mononuclear cells were separated using density gradient centrifugation with the Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and cryopreserved in the CELLBANKER 1 (Nippon Zenyaku Kogyo, Fukushima, Japan). The antibodies used for staining peripheral blood mononuclear cells were as follows; anti-CD4-VioGreen (Miltenyi Biotec, Bergisch Gladbach, Germany); anti-CD3-Paci c Blue/ uorescein isothiocyanate (FITC), anti-CD8-Paci c Blue, anti-CD14-(APC)-Cy7, anti-CD20 allophycocyanin-cyanine 7 (APC-Cy7), anti-CD25 phycoerythrin (PE)-Cy5, anti-CD27-PE-Cy7, anti-CD38-PE-Cy5, anti-CD45RO-PE-Cy7, anti-CD56-PE/PE-Cy7, anti-CD80-FITC, anti-CD86-PE-Cy5, anti-CD127-FITC, anti-CD161-APC and anti-chemokine (C-X-C motif) receptor 3 (CXCR3)-PE (all from BD Biosciences, Franklin Lakes, NJ, USA); anti-CD16-Brilliant Violet 510 and anti-CCR6-Brilliant Violet 421 (both from BioLegend, San Diego, CA, USA); and anti-mouse immunoglobulin G isotypematched controls (VioGreen from Miltenyi Biotec, the others from BD Biosciences). The peripheral cell subsets identi ed have been described in Supplementary table 1.
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