For surface staining, cells were incubated with antibodies diluted in PBS containing 2% FBS for 20 min, on ice. Conjugated antibodies to CD11c (FITC), CD124 (PE), CD14 (PERCP), CD80 (PE-Cy7), CD83 (APC), CD40 (APC-Cy7), CD86 (pacific blue) and HLA-DR (V500) were from BD Biosciences. Conjugated antibodies to CD4 (APC, APC-Cy7 and Alexa-Fluor 488), CD8 (PE and PERCP), CD25 (APC-Cy7) and CD127 (PE-Cy7) were from Biolegend. Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). Cells were stimulated with Leukocyte Activation Cocktail with GoldiPlug (BD Biosciences) for 5 hours at 37°C in 5% CO2, and were then stained with PE anti-IL-4 and APC anti-IFNγ (Biolegend). Regulatory T cells (CD4+CD127−CD25+Foxp3+) were stained for intracellularFoxp3 using the APC anti-human Foxp3 antibody kit (eBioscience). Flow cytometry was performed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ), and theresults were analyzed with FlowJo 8.7 software.
Cd127 pe cy7
Manufactured by BioLegend
Sourced in United Kingdom, United States, Germany
CD127-PE-Cy7 is a fluorescently-labeled antibody that binds to the CD127 (IL-7 receptor alpha chain) antigen. CD127 is expressed on various cell types and is involved in the regulation of immune responses. The PE-Cy7 conjugate provides a bright fluorescent signal for flow cytometry applications.
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Lab products found in correlation
Cd8 bv786, by BD (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Facscanto 2, by BD (2 mentions)
Hla dr v500, by BD (1 mentions)
Leukocyte activation cocktail with goldiplug, by BD (1 mentions)
Apc anti human foxp3 antibody kit, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Il 2 pe, by BD (1 mentions)
Cd44 apc cy7, by BD (1 mentions)
Cd3 bv711, by BioLegend (1 mentions)
Ifn γ pe dazzle594, by BioLegend (1 mentions)
Cd4 buv395, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Fix perm kit, by BD (1 mentions)
Pd 1 pe, by BioLegend (1 mentions)
Ifn γ pe, by BioLegend (1 mentions)
Cd28 bv421, by BioLegend (1 mentions)
Cytoexpert software, by Beckman Coulter (1 mentions)
10 protocols using cd127 pe cy7
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry Staining
3
Comprehensive Immune Cell Phenotyping
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Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8+T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).
4
PBMC Immunophenotyping by Flow Cytometry
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Thawed PBMCs were washed twice in fluorescence-activated cell sorting buffer (PBS containing 0.1% sodium azide and 0.2% bovine serum albumin) and incubated with a viability dye for 15 minutes (Zombie Aqua, BioLegend, San Diego, CA, USA). Cells were incubated for 10 minutes with pooled human immunoglobulin G to block nonspecific antibody binding (FcX solution; BioLegend), and surface staining was performed using fluorescently conjugated antibodies CD3-fluorescein isothiocyanate, CD4-allophycocyanin (APC), CCR4-phycoerythrin (PE), CCR6-Brilliant Violet 605, CD45RO-Brilliant Violet 711, CD25-APC/cyanine 7 (Cy7), and CD127-PE/Cy7 (BioLegend). Flow cytometry data were acquired on an LSRFortessa (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (FlowJo, Ashland, OR, USA).
5
Anti-inflammatory Effects of Nicotine
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MG (purity >98%) and carboxyl methyl cellulose sodium (CMC-Na) were obtained from SanHerb Bioscience (Chengdu, Sichuan, People’s Republic of China) and Aladdin (Shanghai, People’s Republic of China), respectively. Lipopolysaccharide (LPS, from the Gram-negative bacterium E. coli055:B5), nicotine (Nic, α7nAChR agonist), and methyllycaconitine (MLA, α7nAChR specific inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were bought from Hyclone (Logan City, UT, USA). Lymphocyte separation solution was purchased from Solarbio (Beijing, People’s Republic of China). Anti-human CD4-APC, IL-17A-FITC, CD25-FITC, and CD127-PE-CY7 antibodies were obtained from BioLegend (San Diego, CA, USA). ELISA kits (rat TNF-α, IL-1β; human TNF-α, IL-1β, IL-10) were supplied by Boster Biotech (Wuhan, People’s Republic of China). Anti-rat p65, p-p65, and α7nAChR antibodies were products of Cell Signaling Technology (Boston, MA, USA). Biotin-conjugated secondary antibodies were purchased from Beyotime Biotech (Nantong, Jiangsu, People’s Republic of China).
6
Multicolor Flow Cytometry Immunophenotyping
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Intestinal lamina propria cells and MLN cells were labeled with CD4-FITC, CD25-APC (eBioscience), CD8-APC-Cy7, CD3-PerCP (Biolegend), and B220-BV570 (BD Biosciences). Labeling of intracellular FoxP3 was performed after extracellular staining and fixation/permeabilization of cells. GM-CSF-derived dendritic cells were labeled with CD11b-PerCP Cy5.5 (BD Biosciences), CD11c-PE-Cy7, I-A/I-E-APC-Cy7, CD80-PE, CD86-APC, CD8-FITC, and B220-BV570 (Biolegend). Flt3L-derived dendritic cells were labeled with CD11c-PE-Cy7, CD11b- or Siglec-H-PerCP Cy5.5 (Biolegend), I-A/I-E-APC-Cy7, CD317-PE, CD40-Alexa Fluor 647, CD103-FITC, and B220-BV570. Coculture T cells were labeled with CD4-PerCP Cy5.5 (BD Biosciences), CD127-PE-Cy7, CD73-APC, CD195 (CCR5)-FITC, CD62L-BV570, and CD25-APC-Cy7 (Biolegend).
7
Multi-parameter Flow Cytometry Assay
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Anti-Bcl-2 PE, CD3-FITC, CD8-APC, CD127-BV510, IFNγ-APC, RORγt-BV421, TNF-PE were from BD Biosciences. Anti-CD4-PerCP, CD8-BV510, CD127-PECy7, CD161-BV421, -FITC, and -PE, IFNγ-APC, Ki-67-AF488 and -BV510, TNF-BV421, and Vα7.2 PECy7 were from Biolegend. Anti-PLZF APC was from R&D Systems. CellTrace Violet (CTV) cell proliferation kit, Live/Dead Aqua and Near Infrared fixable cell stain were from Life Technologies. MR1-5-OP-RU tetramer -APC, -BV421, and -PE were from NIH Tetramer Core Facility, Emory University. Staining with the MR1 5-OP-RU tetramers was performed for 40 min at room temperature (RT) (6 (link)) before proceeding to the surface and intracellular staining with other mAbs. Cell surface and intracellular staining for TFs, cytokines, and cytotoxic molecules were performed as previously described (10 (link)). Samples were acquired on an FACS Canto II (BD Biosciences) equipped with 405, 488, and 633 nm lasers or CytoFLEX (Beckman Coulter) equipped with 405, 488, and 638 nm lasers. Data including the compensation platform were analysed using FlowJo v.10 (BD Biosciences).
8
Detailed Peptide-Stimulated T Cell Profiling
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Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4+ & CD8+ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO2. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).
9
Multiparameter Flow Cytometry Antibody Panel
10
Flow Cytometry of Memory T Cell Subsets
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Samples of fresh ACD-anticoagulated whole blood were stained with monoclonal antibodies, lysed and fixed, as above. Monoclonal antibodies used to study subsets of CD4 memory T cells were: CD3-PerCP-Cy5.5, CXCR3-Alexa Fluor 488 and -PE-CF594, integrin ß7-APC, CD4-Alexa Fluor 700, CD45RO-FITC, CD45RA-PE-CF594; CD49d-PE, CD25-PE-Cy5, CD8-BV786 (BD Biosciences), CD45RO-ECD (Beckman Coulter); CD49d-BV510, integrin ß7-biotin, CCR6-BV421, CD127-PE-Cy7 (BioLegend); CD161-PE, -APC or PE-Vio-770 (Miltenyi Biotec, Bergisch Gladbach, Germany); and CD62 L-APC-Cy7 (eBiosciences). A total of 400,000 events were collected, and a manual forward and side scatter gate was drawn to include lymphocytes, then gated sequentially on CD31, on CD41, and then on CD45RO1 cells using FlowJo, then exported as an FCS file using FlowJo.
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