Human colorectal adenocarcinoma epithelial cells, Caco-2 cells (HTB-37), were purchased from American Type Culture Collection (ATCC, Manassas, VA), and cultured in ATCC-formulated Eagle’s Minimum Essential Media (30-2003, ATCC, Manassas, VA) supplemented with 20% foetal bovine serum (FBS, F2442, Sigma-Aldrich, St. Louis, MO) at 37 °C with 5% CO2. GP (HY-N6881, C41H70O12, purity: ≥90.0%, CAS no. 80325-22-0, MedChemExpress, Monmouth Junction, NJ) were solubilized in dimethyl sulphoxide (D2650, DMSO, Sigma-Aldrich, St. Louis, MO), and diluted into the gradient concentrations of 0, 50, 100, 150 or 200 μM for the use in drug treatment. Notably, DMSO was set at the 0.1% final concentration in in vitro experiments. Also, gradient GP were applied in the measurement of cell viability. Then, Caco-2 cells were assigned into four groups (control group, LPS group, LPS + GP150 group and LPS + GP200 group). Caco-2 cells in the LPS + GP150 group or the LPS + GP200 group were treated with 150 μM or 200 μM of GP at 37 °C for 24 h with 5% CO2. After being washed with phosphate-buffered saline (PBS, P5493, Sigma-Aldrich, St. Louis, MO), cells were exposed to 10 μg/mL LPS (HY-D1056, LPS, MedChemExpress, Monmouth Junction, NJ) at 37 °C for 24 h with 5% CO2 (He et al. 2020 (link)). Cells in the LPS group only underwent 10 μg/mL LPS exposure for 24 h, and cells in the Control group remained untreated.
Hy d1056
Manufactured by MedChemExpress
Sourced in United States
HY-D1056 is a laboratory instrument designed for the purification and analysis of various biomolecules, such as proteins, nucleic acids, and other macromolecules. The core function of this equipment is to perform chromatographic separation techniques, allowing researchers to isolate and characterize target analytes from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Hy 18739, by MedChemExpress (2 mentions)
Caco 2 cells htb 37, by American Type Culture Collection (1 mentions)
D2650, by Merck Group (1 mentions)
P5493, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
M6250, by Merck Group (1 mentions)
Ifn γ, by Novoprotein (1 mentions)
Il 13, by Novoprotein (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
6 protocols using hy d1056
1
Caco-2 Cell Viability Assay with LPS and Glycyrrhizic Acid
2
Mouse Alveolar Macrophage Polarization Assay
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Mouse alveolar macrophages MH-S (iCell-m078, iCell Bioscience, Shanghai, China) were cultured in complete medium containing DMEM, 10% fetal bovine serum (13011-8611, Every Green, Taiwan), 0.05 mM β-mercaptoethanol (M6250, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (C0222, Beyotime, Suzhou, China). In addition, Lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA) with sh-S100A8 or sh-S100A9 was incubated with cells (2 × 105 cells/well) in 6-well plates for 48 h. Furthermore, macrophage polarization was induced for 8 h with OVA (Wang et al., 2021 (link)). Lipopolysaccharide (LPS) (100 ng/mL, HY-D1056, MedChemExpress, Boston, MA, USA) as a TLR4 agonist was incubated with MH-S cells for 8 h (Alhouayek et al., 2013 (link)) with PBS as a control. The cells were divided into Control, OVA, OVA + sh-S100A8, OVA + sh-S100A9, OVA + sh-S100A8 + LPS, and OVA + sh-S100A9 + LPS groups. Cell experiments of cells were repeated independently at least thrice.
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
RNA-seq Analysis of Macrophage Polarization
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RAW 264.7 macrophages purchased from the Cell Resource Center of Shanghai Institute for Biological Sciences were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (FBS, ScienCell Research Laboratories, Carlsbad, CA, USA), 1%penicillin and 1% streptomycin (HyClone). Cells were incubated under standard conditions with constant temperature (37° C) and an atmosphere of 5% CO2. Cells were grown to 60–80% confluence before use. In order to perform RNA-seq, cells were treated for 12 h with RU.521 (cGAS inhibitor 2 μg/mL, InvivoGen, San Diego, CA, USA) or with DMSO as vehicle control. In addition, RAW264.7 macrophages were pretreated with RU.521 (2 μg/mL) for 12 h and then incubated for another 6 h with LPS (10 ng/mL, HY-D1056, MedChemExpress, Monmouth Junction, NJ, USA) plus IFNγ (20 ng/mL, HY-P7071, MedChemExpress) that polarize macrophages to M1.
5
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.
6
Macrophage Polarization and Fibroblast Migration
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RAW264.7 cells were stimulated with 100 ng/mL lipopolysaccharides (LPS, #HY-D1056, MedChemExpress, China) for 24 h, and cocultured with 100 µg/mL of MVs or Smad7-MVs for 24 h at 37℃ and 5% CO 2 . Cells were treated with equal volume of PBS as controls. The levels of IL-6, IL-1β and TNF-α in cell supernatants were measured using enzyme-linked immunosorbent assay (ELISA), and markers of M1 macrophages, CD86 and iNOS, were measured using flow cytometry and immunofluorescence.
Fibroblasts were stimulated with 10 ng/mL TGF-β1 (#HY-P7118, MedChemExpress, China) for 24 h, and cocultured with 100 µg/mL of MVs or Smad7-MVs for 24 h. Cells were treated with equal volume of PBS as controls. Migration of cells was measured using Transwell. The markers of fibroblast differentiation, α-smooth muscle actin (SMA) and collagen III, were measured using immunofluorescence and western blotting.
Fibroblasts were stimulated with 10 ng/mL TGF-β1 (#HY-P7118, MedChemExpress, China) for 24 h, and cocultured with 100 µg/mL of MVs or Smad7-MVs for 24 h. Cells were treated with equal volume of PBS as controls. Migration of cells was measured using Transwell. The markers of fibroblast differentiation, α-smooth muscle actin (SMA) and collagen III, were measured using immunofluorescence and western blotting.
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