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6 protocols using thp 1 cell line

1

Cell Line Cultivation and Polarization Protocols

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HCC SK-HEP-1, HEK 293T and human acute monocytic leukemia cell line THP-1 were purchased from the American Type Culture Collection (ATCC). HA22T cell line (BCRC No. 60168) was gifted from Prof. Yuh-Shan Jou, Academia Sinica, Taiwan. HA22T, SK-HEP-1 and HEK 293T cell lines were cultured in DMEM (Invitrogen) with 10% Fetal Bovine Serum, 1% penicillin/streptomycin and 1% glutamine. THP-1 cell line was cultured in RPMI1640, 100 ng/ml Phorbol 12-myristate 13-acetate (PMA, Sigma) was used for 48 hours to induce THP-1 differentiation into macrophages. To induce M2 polarized phenotype, 20 ng/ml of IL-4(Invitrogen) and IL-13 (R&D Systems) were used for 48 hours. All cell lines were cultured in a 5% (v/v) CO2 humidified incubator at 37 °C.
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2

Evaluating Mg Antioxidant Capacity

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Human monocytes (THP-1 cell line, Cat#88081201; Sigma-Aldrich, St Louis, MO, USA) were cultured in RPMI-1640 medium with 1% penicillin–streptomycin (Basel, Switzerland) and 10% fetal bovine serum (FBS, Sigma-Aldrich).
All different assays were performed under basal and stimulating pro-inflammatory conditions, such as those induced by bacterial lipopolysaccharide (LPS), to assess whether the antioxidant capacity of Mg could protect patients from potential high-risk situations.
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3

Generating M0, M1 and M2 Macrophages from THP-1 Cells

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The human monocyte THP-1 cell line was obtained from ATCC (American Type Culture Collection) and cultured in RPMI culture medium (Sigma) supplemented with 10% FBS and 1% P/S at 37 °C in a humified 5% CO 2 atmosphere. Subculturing was performed routinely before the cell density reached 1 × 10 6 cells/ml. Differentiation of monocytes towards naïve macrophages (M0) was induced by stimulating with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 3 days, followed by 24 h cultivation with PMA-free fresh RPMI medium [72, (link)73] (link). The M1 phenotype was chemically provoked by stimulating the naïve macrophages with 20 ng Interferon-γ (IFN-γ; Miltenyi Biotec) and 100 ng LPS (Lipopolysaccharide; Sigma), while the M2 phenotype was triggered by 20 ng Interleukin-4 (IL-4; Miltenyi Biotec) stimulation for 24 h. The validation of a successful chemical polarization was performed by measuring specific surface markers CCR7 (M1) and MRC1 (M2) on the gene expression and protein level. For experiments, naïve (M0) macrophages were cultured on collagen coated tissue culture plastic (TCP) or electrospun PCL nanofiber substrates at a density of 100,000 cells/cm 2 and submerged in 3 ml of culture medium.
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4

Culturing Human Monocytic Leukemia Cells

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The THP-1 cell line (ECACC, Salisbury, UK) was an immortalized, human monocytic leukemia derived cell line grown in RPMI 1640 medium (Sigma Aldrich-MERCK) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 2 mMol/L-glutamine (Sigma–Aldrich–MERCK, St. Louis, MO, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Sigma–Aldrich–MERCK, St. Louis, MO, USA) and kept at 37 °C, 5% CO2 in a humidified tissue culture incubator.
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5

Monocyte Polarization Protocol

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Isolated human monocytes were allowed to rest for 24 h prior to stimulation in RPMI 1640 Medium (Gibco, ThermoFisher, Paisley, UK) supplemented with 1% L-glutamine (Gibco), 1% penicillin/streptomycin (Gibco) and 5% de-complemented human serum.
THP-1 cell line (Sigma–Aldrich, St-Louis, MO, USA), a human monocytic cell line derived from monocytic leukemia patients, was cultured in RPMI 1640 supplemented with L-glutamine, 10% fetal calf serum (FCS; VWR International Ltd, Lutterworth, UK) and 1% penicillin and streptomycin (PenStrep) at 37 °C with 5% CO2.
THP-1 or monocytes were seeded into 24-well plates (6 × 105cells/mL) in RPMI 1640 and were left untreated or were treated with 20 ng/mL interleukin-4 (IL-4; Fischer Scientific, Loughborough, UK), 20 ng/mL interleukin-10 (IL-10; PeproTech, London, UK) or 100 ng/mL lipopolysaccharide (LPS; Sigma–Aldrich) for 24 h or in some experiments for the time of 48 h. Cells were washed following stimulation to ensure serum was removed.
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6

Cell Culture Protocol for THP-1, BICR-18, and CAPAN-2

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The human pro-monocytic THP-1 cell line was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cells were grown at 37 °C in suspension in RPMI 1640 medium GlutaMAX™ (Fisher Scientific; Madrid, Spain) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. THP-1 cells were plated at a density of 3 × 105 in 24-well culture plates and differentiated to macrophages through a first incubation with 100 nM phorbol 12-myristate13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The PMA-containing media was discarded and replaced with fresh media without PMA for a further 24 h.
The human epithelial cell line BICR-18 (from larynx squamous cell carcinoma) and CAPAN-2 (from pancreatic ductal adenocarcinoma) were obtained from American Type Cell Collection (ATCC, Manassas, VA, USA) and maintained at 37 °C in DMEM GlutaMAX™ (Fisher Scientific; Madrid, Spain) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were split every 3 days by trypsinization with 0.1% trypsin in Ca2+/Mg2+-free phosphate-buffered saline (PBS) containing 0.9 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich; St. Louis, MO, USA).
All cells were cultured at 37 °C in humidified atmosphere of 95% air and 5% CO2.
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