Whole blood and fresh tumor samples from diagnostic surgery were processed within 12 hours of collection. Ten samples from patients with benign pleural pathologies of infectious and inflammatory nature were also included as a comparison. Whole blood was either lysed using ammonium chloride solution according to the manufacturer’s instructions (Qiagen) or using a hypertonic ammonium chloride solution (150 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1mmol/L EDTA) for 10 minutes at room temperature (maintained at 21°C −23°C) at a ratio of 1:9 (volume of sample: volume of lysing solution) prior to antibody staining. Where indicated peripheral blood was separated using a Lymphoprep density gradient. Tissue samples were digested using type II collagenase (Worthington) for 3 hours at 37°C. Immune populations were identified by staining with anti-CD11b, anti-HLA-DR, anti-CD13, anti-CD14, anti-CD15, anti-CD66b, and anti CD45 antibodies (BD Biosciences) on ice or at room temperature for 30 minutes. Cells were acquired using FACS-Canto II (BD Biosciences) and Cyan (Beckman Coulter) and analyzed either by FCS Express 4 software (DeNovo Software) or FlowJo (TreeStar).
Anti cd66b
Manufactured by BD
Sourced in Japan, United States
Anti-CD66b is a monoclonal antibody that specifically binds to the CD66b antigen, which is expressed on the surface of human granulocytes, including neutrophils, eosinophils, and basophils. This antibody can be used for the identification and enumeration of these cell types in various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b, by BD (4 mentions)
N formyl methionyl leucyl phenylalanine, by Merck Group (2 mentions)
Wkymvm peptide, by Merck Group (2 mentions)
Anti cd35, by BD (2 mentions)
Anti cd63, by BD (2 mentions)
Tumor necrosis factor α, by Merck Group (2 mentions)
Anti cd62l, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
Fcs express 4, by De Novo Software (1 mentions)
Anti cd15, by BD (1 mentions)
Anti hla dr, by BD (1 mentions)
Anti cd45 antibody, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Ab25377, by Abcam (1 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
Af3870, by R&D Systems (1 mentions)
Sp 8400, by Vector Laboratories (1 mentions)
Nel744b001kt, by PerkinElmer (1 mentions)
Human serum, by Merck Group (1 mentions)
8 protocols using anti cd66b
1
Immunophenotyping of Pleural Samples
2
Immunohistochemical Analysis of Lung Tissue
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Murine paraffin-embedded blocks were prepared from lungs fixed via the trachea with 10% buffered formalin at 20 cm H2O. Serial sections were stained with anti-Ly6G clone 1A8 (ab25377, Abcam) and the following polyclonal antibodies: anti-SEMA3F (SAB2700501, Sigma-Aldrich), NRP1 (AF3870, R&D Systems), NRP2 (H300, Santa Cruz Biotechnology), or an isotype control following deparaffinization. Lung sections from patients with COPD undergoing resection for suspected lung tumor were stained with anti-SEMA3F, NRP2, or isotype control, and visualized with DAB or stained with anti-CD66b (555723, BD Biosciences), anti-NRP2 (HPA054974, Atlas Antibodies), and DAPI (422801, Sigma-Aldrich) with TSA plus system amplification (NEL744B001KT, Perkin Elmer) and autofluorescence quenching with TrueView (Vector, SP-8400).
3
Single-cell sorting for MP and CD45+ analysis
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Single-cell suspensions were then preincubated with a blocking solution containing 1% human serum (Sigma-Aldrich, catalog no. H3667) in saline solution and stained with fluorophore-conjugated primary antibodies at room temperature in the dark for 15 minutes: anti-CD45 (BD Biosciences, clone HI30, RRID:AB_1645452), anti-CD66b (BD Biosciences, clone G10F5, RRID:AB_396067), and anti-CD163 (BD Biosciences, clone GHI/61, RRID:AB_2737697). Cell viability was assessed using Zombie Aqua Fixable Viability Kit (BioLegend, catalog no. 423101). MP and CD45+ cells were sorted on a FACSAria III (BD Biosciences) as shown in Supplementary Fig. S1B.
4
Neutrophil Activation and Degranulation Assay
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Peripheral blood neutrophils were isolated as recorded previously [27] . After isolation, granulocytes were kept on ice in KRG buffer (pH 7.3; 120 mM NaCl, 5 mM KCl, 1.7 mM KH 2 PO 4 , 8.3 mM NaH 2 PO 4 , 10 mM glucose), supplemented with Ca 2+ (1 mM) and Mg 2+ (1.5 mM). For assessment of neutrophil activation and degranulation, cells were left untreated at room temperature or incubated for 20 min at 37℃ with or without N-Formylmethionyl-leucyl-phenylalanine (fMLP) (50 nM; Sigma-Aldrich), tumor necrosis factor α (10 ng/mL; Sigma-Aldrich), or WKYMVM peptide (50 nM; Sigma-Aldrich). Granulocytes were xed and erythrocytes lysed by treatment with FACS lysing solution (BD Biosciences) for 15 min at 4℃. Cells were then stained with anti-CD11b, anti-CD62L, anti-CD35, anti-CD63, or anti-CD66b antibodies (all from BD Biosciences) for 1 h on ice. A minimum of 10,000 neutrophils were assessed by ow cytometry [28] .
5
Emodin Modulates Neutrophil Activity
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Emodin (purity > 98%) was purchased from Xi-an Helin Biological Engineering Co. (Xi-an, Shanxi, China). Urethane, phorbol 12-myristate 13-acetate (PMA), 4′,6-diamidino-2-phenylindole (DAPI) and 2′,7′-dichlorodihydrofluorescein (DCFH-DA) diacetate were purchased from Sigma Chemical Co (St.Louis. MO. USA). Ly6G microbeads, CD66b microbeads and Ly6G monoclonal antibody (Ly6G mAb) were from Miltenyi. Antibodies including anti-CD66b, anti-histone Cit-H3, anti-PAD4, FITC-conjugated anti-mouse CD66b were obtained from BD Pharmingen. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG polyclonal antibody, peroxidase substrate DAB (3,3′-diaminobenzidine) were obtained from Nichirei Bioscience (Tokyo, Japan). Annexin V-FITC Apoptosis kit and mouse quantitative ELISA kits (IFN-γ, ROS, IL-12, TNF-α, IL-6 and TGF-β1) were obtained from R&D Systems. Four coagulation kits were obtained from Nanjing jiancheng bioengineering institute. Standard rodent chow was purchased from Henan Provincial Medical Laboratory Animal Center (Zhengzhou, China), License No. SCXK (YU) 2015-0005, Certificate No. 41000100002406.
6
Neutrophil Activation and Phenotyping
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Isolated neutrophils were activated for 30 min at 37°C with ionomycin (IO; nal concentrations: 1 µM and 4 µM; Thermo Fisher) and phorbol 12-myristate 13-acetate (PMA; nal concentrations: 100nM and 600nM; Sigma Aldrich). Afterwards, samples were xed with paraformaldehyde (PFA; nal concentration: 1%, Thermo Fisher) and stained for 15 min, RT in the dark with the following antibodies: anti-CD66b (1:4), anti-CD11b (1:4) and propidium iodide (PI, 1:4; BD Bioscience). PBS (Gibco) was added to each sample to stop the staining procedure. All samples were analysed using a CytoFLEX S instrument (Beckman Coulter). For analysis, median uorescent intensity (MFI) of CD66b and CD11b of live, single CD66b+ cells were investigated. Furthermore, "CD11b/CD66b high" neutrophils were de ned by setting the respective immediately xed samples as baseline. Data analysis was performed using FlowJo V10 (FlowJo, LLC).
7
Neutrophil Activation and Degranulation Assay
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Peripheral blood neutrophils were isolated as recorded previously [27] . After isolation, granulocytes were kept on ice in KRG buffer (pH 7.3; 120 mM NaCl, 5 mM KCl, 1.7 mM KH 2 PO 4 , 8.3 mM NaH 2 PO 4 , 10 mM glucose), supplemented with Ca 2+ (1 mM) and Mg 2+ (1.5 mM). For assessment of neutrophil activation and degranulation, cells were left untreated at room temperature or incubated for 20 min at 37℃ with or without N-Formylmethionyl-leucyl-phenylalanine (fMLP) (50 nM; Sigma-Aldrich), tumor necrosis factor α (10 ng/mL; Sigma-Aldrich), or WKYMVM peptide (50 nM; Sigma-Aldrich). Granulocytes were xed and erythrocytes lysed by treatment with FACS lysing solution (BD Biosciences) for 15 min at 4℃. Cells were then stained with anti-CD11b, anti-CD62L, anti-CD35, anti-CD63, or anti-CD66b antibodies (all from BD Biosciences) for 1 h on ice. A minimum of 10,000 neutrophils were assessed by ow cytometry [28] .
8
Quantifying Cell-Derived Extracellular Vesicles
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Total MV concentration was determined by taking advantage of the MV ubiquitous Phtdser exposure (37) . MVs from PPP were captured onto insolubilized biotinylated annexin-5, a protein with high affinity for Phtdser, using streptavidin-coated microtitration plates (Roche Diagnostics, Deutschland). After three washings, MVs were measured by prothrombinase assay in which blood clotting factors (FXa, FVa, FII) and calcium concentrations ensure that the MV PhtdSer is the rate-limiting parameter of the reaction (Supp methods) (37) . To determine their cell origin, MVs from PPP were first washed by two centrifugation steps (60 min, 14.000g, 4°C), concentrated in 500µl before capture onto biotinylated antibodies against T-helper cells (anti-CD4), cytotoxic T-cells (anti-CD8), B-lymphocytes (anti-CD19), monocytes (anti-CD14) (all from Leinco, St Louis, Missouri, USA), activated neutrophils (anti-CD66b; BD Bioscience, New Jersey, USA) before prothrombinase assay. The MV concentration was obtained for each cell origin by subtracting the OD values measured using the isotype biotinylated control immunoglobulin (BD Bioscience).
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