Isolated neutrophils were plated at a density of 9×106 cells per well in a 6-well plate. Then, 500 nM PMA (HY-18739, MedChemExpress) was added to each well and incubated in a 37 °C, 5% CO2 incubator for 3 h 40 (link). The samples were collected by scraping the cells and transferred to new centrifuge tubes, which were then centrifuged at 450 g and 4 °C for 10 min. The resulting supernatant contained NETs, whose concentration was measured using the Quant-iT Picogreen dsDNA Assay Kit (P11496, Thermo Fisher). NETs were confirmed by staining with SYTOX Orange (S11368, Thermo Fisher) and visualized using confocal microscopy (LSM 880, Zeiss).
Hy 18739
Manufactured by MedChemExpress
Sourced in United States
HY-18739 is a laboratory equipment product offered by MedChemExpress. It is a device designed to assist in scientific research and experiments. The core function of HY-18739 is to perform a specific task or analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Hy d1056, by MedChemExpress (2 mentions)
Lsm 880, by Zeiss (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Novoprotein (1 mentions)
Il 13, by Novoprotein (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
Mab2263, by Merck Group (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Ab5847, by Merck Group (1 mentions)
Cd11b apc, by BioLegend (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Pe sirpα, by BioLegend (1 mentions)
St476, by Beyotime (1 mentions)
Rpmi 1640, by Wuhan Servicebio Technology (1 mentions)
Fsp500, by ExCell Bio (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Thp 1 cell, by American Type Culture Collection (1 mentions)
10 protocols using hy 18739
1
Neutrophil Extracellular Trap Quantification
2
Isolation and Quantification of Neutrophil Extracellular Traps
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Purified neutrophils were inoculated in a six-well plate (1 × 106 cells/well), stimulated with 100 nM phorbol myristate acetate (HY-18739; MedChemExpress LLC, USA), and cultured in a 5% CO2 incubator for 4 h at 37 °C. The upper medium was then gently suctioned, leaving NETs and neutrophils. Precooled PBS without calcium and magnesium was added, and the NETs and neutrophils attached to the bottom layer were eluted. The liquid from the six-well plate was collected and centrifuged at 450 × g for 10 min at 4 °C, and the supernatant was collected and centrifuged at 15,000 × g for 15 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in PBS. The concentration was determined using a micro-DNA instrument (206–26,300-48; BioSpec-nano, Japan), and the sample was stored at -20 °C.
3
Macrophage Polarization Protocol
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M0 macrophages were obtained by treating THP-1 cells with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, HY-18739, MedChemExpress, USA) for 48 h. Then, M0 macrophages were induced with 100 ng/ml bacterial lipopolysaccharide (LPS, HY-D1056, MedChemExpress, USA) + 2.5 ng/ml IFN-γ (C014, novoprotein, China) for 48 h to acquire the M1 phenotype. Meanwhile, M0 macrophages were induced with 10 ng/mL IL-4 (CX03, novoprotein, China) + 10 ng/mL IL-13 (CC89, novoprotein, China) for 48 h to acquire the M2 phenotype. Polarized status was confirmed utilizing flow cytometry.
4
Macrophage Polarization and Hydrogen Treatment
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The THP-1 cell suspension was spread evenly in 48-well plates at 1 × 105 cells/well. M0 macrophages were obtained by treating THP-1 cells with PMA (MedChemExpress, HY-18739) (100 ng/mL) for 48 h. Then, M0 macrophages were induced with LPS (MedChemExpress, HY-D1056) (100 ng/mL) and IFN-γ (Novoprotein, CI57) (20 ng/mL) for 48 h in the presence of PMA to acquire the M1 phenotype. Meanwhile, M0 macrophages were induced with IL-4 (Novoprotein, CX03) (20 ng/mL) and IL-13 (Novoprotein, CC89) (20 ng/mL) for 48 h to acquire the M2 phenotype. For the H2 group, H2-rich PRIM1640 medium was added into each well with M0 macrophages.
5
Quantifying PMA-Induced GluR1 Phosphorylation
6
Sericin Modulation of U937 Cell Polarization
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The capability of the sericin to influence polarization in the U937 cell was evaluated. U937 cells at a density of 6 × 106 cells/well were incubated in a complete RPMI-1640 medium for 24 h at 37°C in 5% CO2. Cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Cat. No.: HY-18739, MedChemExpress, United States) for macrophage activation, and then treated with 0.1 and 1.0 mg/ml of sericin, and 10 ng/ml of LPS (Escherichia coli 055: B5, Cat. No.: HY-D1056, MedChemExpress, United States), respectively. The cells were incubated for 24 h. The medium was removed before treatment with 1 μg/ml of LPS, and cells were washed with 5 ml of PBS and replenished with a complete medium. Cells treated with 10 ng/ml LPS alone were used as the control. After incubation, the cells were washed twice with PBS and resuspended in 0.5 ml of staining buffer PBS containing 1% FBS and 0.09% (w/v) sodium azide.
7
Isolation of Cell-free NETs from GC Patients
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Cell-free NETs were isolated from neutrophils of GC patients as previously described, with modifications[38 ]. Neutrophils (107 cells/mL) were cultured for 4 h at 37°C under 5% CO2 in medium supplemented with 500 nM PMA (HY-18739; MedChemExpress, Monmouth Junction, NJ, USA). The supernatant was discarded, and ice-cold 1 × PBS was added to wash down the cell layer of neutrophils to obtain the NET medium and centrifuged at 1500 g for 10 min at 4°C to remove cell debris. Thereafter, 1.5 mL supernatant (sterile DNA–protein complex) was centrifuged at 15 000 g for 15 min at 4°C. The resultant pellets were suspended in ice-cold 1 × PBS, followed by DNA concentration measurement in the medium obtained using spectrophotometry (Biospec-nano, Japan). An adequate DNA concentration in the medium should range between 50 and 100 μg/mL. The medium containing the NETs was stored at -80°C for subsequent experiments.
8
Morphine Effects on Macrophage Immunophenotype
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THP-1 cells (5 × 105 cells/well) were seeded in 12-well plates and cultured with 200 ng/mL of phorbol-12-myristate-13-acetate (PMA, HY-18739 MedChemExpress, South Brunswick Township, NJ, USA) for 48 h to differentiate into macrophages [54 (link)]. THP-1-derived macrophages were exposed to different concentrations of morphine for 48 h. The supernatant was then removed and the cells were digested with 0.25% trypsin (25200-072, Gibco, NY, USA). After washing, the cells were resuspended in 100 μL of PBS (ST476, Beyotime, Shanghai, China). For surface staining, cells were labeled with APC-CD11b (101212, Biolegend, CA, USA), PE-SIRPα (372103, Biolegend, CA, USA), and APC-CD47 (17-0479-42, eBiosciences, CA, USA). Cell suspensions were incubated with appropriate antibodies for 30 min at room temperature in the dark, followed by a washing step to remove unlabeled antibodies. Flow cytometry analysis was performed using BD LSRFortessa™ (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FlowJo 10.6 software.
9
Differentiation of THP-1 Cells into Foam Cells
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Human THP-1 cells (CL-0233) were kindly provided by Procell Life Science&Technology (Wuhan, China) and subjected to STR authentication in Procell Life Science&Technology. THP-1 cells were cultured with RPMI-1640 (G4530-500ML, Servicebio, China) supplemented with 10% Fetal Bovine Serum (FBS) (FSP500, ExCell Bio, China) and 1% Penicillin-Streptomycin (P/S) (15,140,122, GIBCO, USA). The cells were inoculated in 6-well plates at a density of 5 * 10^5/ml, and the cells were inoculated with phorbol 12-myristate 13-acetate (PMA) (100 ng/ml; Hy-18,739, MedChemExpress, USA) was cultured in the culture medium for 48 h to differentiate into macrophages. THP-1 macrophages differentiated from PMA were treated with 80 µg/ml ox-LDL (Yiyuan Biotechnology, China) for 24 h. Foam cell development was determined using Oil Red O staining. An incubator with 5% CO2 and 37 °C was used to culture cells.
10
Differentiation of THP-1 cells into Macrophages
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THP-1 cells (human acute monocytic leukemia cell line) from the American Type Culture Collection (ATCC, USA) were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 0.05 mM 2-mercaptoethanol at 37 • C in a 5% CO 2 incubator. After 72 h, macrophages were obtained from the THP-1 cells in RPMI-1640 medium (Gibco by Life Technologies, Grand Island, NY) supplemented with PMA (HY-18739, 80 nM, Med Chem Express, China).
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