Cells were cultured until confluence and then wounded using a 200 µl yellow pipette tip. Cells were treated with Oncostatin M (OSM) (R&D systems) at 100–200 ng/mL every 48 h during 4 days. Three wounds were made for each sample, and migration distance was photographed and measured at time 0 h and every 12 h until 48 h.
Oncostatin m osm
Manufactured by R&D Systems
Sourced in United States
Oncostatin M (OSM) is a cytokine that belongs to the interleukin-6 family. It is involved in various cellular processes, including cell growth, differentiation, and inflammation. OSM can be used in research to study its biological functions and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
B27 supplement minus vitamin a, by Thermo Fisher Scientific (2 mentions)
Glutamax, by R&D Systems (2 mentions)
B27 supplement minus vitamin a, by R&D Systems (2 mentions)
Activin a, by R&D Systems (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Retinoic acid, by Merck Group (2 mentions)
Periodic acid schiff staining kit, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps from pseudomonas aeruginosa, by Merck Group (1 mentions)
5 aza 2 deoxycytidine 5 aza, by Merck Group (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
M csf, by R&D Systems (1 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (1 mentions)
16 protocols using oncostatin m osm
1
Wound Healing Assay with Oncostatin M
2
Hepatic and Bile Duct Differentiation Protocol
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Hepatocyte differentiation was induced by switching the medium to basal SCMA supplemented with 20 ng/ml HGF (R&D system), 20 ng/ml Oncostatin M (OSM, R&D system), 0.1 μM Dexamethasone (Dex, Sigma-Aldrich), and with 2 μM TGFβ receptor inhibitor (E-616452) or γ-secretase inhibitor (Compound E, 0.5 and 1 μM) for 2 weeks. The hepatic differentiation was verificated by glycogen storage assay using a periodic acid-Schiff staining kit (Sigma-Aldrich) according to the product instructions. Bile duct differentiation was induced by 3D culture. iHepSCs were suspended in cold SCMA supplemented with 20 ng/ml EGF and mixed 1 : 1 with Matrigel. Then, the mixture was plated into 24-well plate (0.5 ml/well) and placed in an incubator at 37°C for 30 min to allow the formation of 3D Matrix. 0.5 ml of SCMA was then carefully overlayed on the gel. The cells were cultured for approximately 1 week to allow the formation of bile duct-like structures [16 (link)].
3
Macrophage Polarization and Regulation
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Monocytes were isolated from buffy coats of healthy blood donors (Sanquin Blood Bank, Leiden, The Netherlands) using anti-CD14 microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s protocol. MΦ1 and MΦ2 were derived as described previously [23 (link), 24 (link)]. Briefly, cells were cultured for six days in RPMI 1640 medium in 48 well plates (Invitrogen, Life Technologies, Bleiswijk, The Netherlands) containing 10 % fetal calf serum (FCS, Invitrogen), 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin (all Bio Whittaker, Walkersville, MD, USA) at 37 °C in 5 % CO2 atmosphere with either GM-CSF (5 ng/ml, Invitrogen) or M-CSF (50 ng/ml, R&D Systems, Minneapolis, MN, USA) to obtain MΦ1 and MΦ2, respectively [13 (link), 14 (link)]. Differentiated macrophages were stimulated with the pro-inflammatory stimuli lipopolysaccharide (LPS, from Pseudomonas aeruginosa, 100 ng/ml, Sigma-Aldrich, St. Louis, MO, USA), TNF-α (10 ng/ml, Peprotech, Rocky Hill, NJ, USA) or oncostatin M (OSM, 100 ng/ml, R&D Systems) for 24 h. Dexamethasone (0.1, 0.3 and 1nM, Sigma) was added during differentiation at day 0, 3 and/or day 7. The demethylating agent 5-AZA-2’-deoxycytidine (5-AZA, 0.1, 1 and 10 μM, Sigma) was added during differentiation. Every day 100 μl per well was removed and replaced by fresh medium containing growth factors and 5-AZA.
4
Differentiation of Dlk+ Cells
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Dlk+ cells (1 × 103 cells/well) were plated on collagen type IV-coated 6-well plates (Becton Dickinson, Franklin Lakes, NJ) and cultured as described elsewhere [13 (link)]. At least three independent experiments were performed for colony assays. To evaluate the ability to differentiate into hepatocytes, Dlk+ cells were placed on an Engelbreth-Holm-Swarm (EHS) gel (Becton Dickinson) in the presence of oncostatin M (OSM, R&D Systems, Minneapolis, MN). Similarly, the cells were placed on collagen type I gel (Nitta Gelatin, Osaka, Japan) in the presence of tumor necrosis factor- (TNF-) α (PeproTech, Rocky Hill, NJ) to examine their potential to differentiate into cholangiocytes.
5
Differentiation of Human iPSCs into Hepatocytes
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Human iPS cells were dissociated into single cells with TrypLE Select Enzyme and seeded on Matrigel (growth factor reduced; Corning, NY, USA)-coated plates. Differentiation of human iPS cells into hepatocytes was performed based on our previous methods with some modifications.20 (link) Briefly, in the first step, human iPS cells were cultured in RPMI1640 medium (Sigma-Aldrich) with 1x GlutaMAX (Thermo Fisher Scientific), 0.5x B27 Supplement Minus Vitamin A (Thermo Fisher Scientific), and 100 ng/mL Activin A (R&D Systems, MN, USA) for 4 days to induce definitive endoderm cells. In the second step, definitive endoderm cells were cultured in RPMI1640 medium with 1x GlutaMAX, 0.5x B27 Supplement Minus Vitamin A, 20 ng/mL bone morphogenetic protein (BMP) 4 (R&D Systems) and 20 ng/mL fibroblast growth factor (FGF) 4 (R&D Systems) for 5 days to induce hepatoblast-like cells. In the third step, hepatoblast-like cells were cultured in RPMI1640 medium with 1x GlutaMAX, 0.5x B27 Supplement Minus Vitamin A, and 20 ng/mL hepatocyte growth factor (HGF; R&D Systems) for 5 days to HLCs. In the fourth step, the cells were cultured in hepatocyte culture medium (HCM; Lonza, Basel, Switzerland) containing 20 ng/mL oncostatin M (OsM; R&D Systems) without epidermal growth factor for 11 days for maturation.
6
Arsenite and Antimonite Regulation of SIK
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SIK cultures were treated at confluence with 50 ng/ml Oncostatin M (OSM) (R&D Systems), 3 µM sodium arsenite, 6 µM potassium antimony tartrate (antimonite) or not treated. In some experiments cell cultures were pretreated for 30 min with 10 µM U1026 (an inhibitor of MEK1 and MEK2) (LC Laboratories) or 1 µM JAK Inhibitor I (Calbiochem) before addition of OSM, arsenite or antimonite. To test the influence of hydrocortisone on arsenite or antimonite effects, SIK cultures were grown to confluence in normal culture medium, then switched to medium containing serum depleted of steroids by charcoal/dextran treatment60 in the presence and absence of combinations of 10 µM hydrocortisone, 3 µM arsenite and 6 µM antimonite. RNA was prepared using Trizol reagent (Life Technologies), followed by cDNA preparation using an Applied Biosystems reverse transcription kit. cDNA was analyzed by qPCR with Taqman assays (Applied Biosystems), normalizing to MAPK1, a gene product demonstrated by transcriptomic analysis not to be changed by arsenite and antimonite treatments.
7
Hepatic Liver Organoid Differentiation
8
Stepwise Differentiation of hiPSCs to Hepatocytes
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Before the initiation of hepatic differentiation, human iPS cells were dissociated into single cells by using TrypLE Select Enzyme and plated onto Matrigel-coated dishes. The cells were then cultured in StemFit AK02N medium for 24 hours. The differentiation protocol for the induction of definitive endoderm cells, hepatoblast-like cells, and HLCs was based on our previous reports [4 (link)] with some modifications. Briefly, in the definitive endoderm differentiation, human iPS cells were cultured for 4 days in RPMI1640 medium (Sigma-Aldrich), which contained 100 ng/mL Activin A (R&D Systems), 2× GlutaMAX (Thermo Fisher Scientific), and 0.5× B27 Supplement Minus Vitamin A (Thermo Fisher Scientific). For the induction of hepatoblast-like cells, the definitive endoderm cells were cultured for 5 days in RPMI1640 medium containing 20 ng/mL BMP4 (R&D Systems), 20 ng/mL fibroblast growth factor 4 (FGF4; R&D Systems), 2× GlutaMAX, and 0.5× B27 Supplement Minus Vitamin A. To perform hepatic differentiation, the hepatoblast-like cells were cultured for 5 days in RPMI1640 medium containing 20 ng/mL hepatocyte growth factor, 2× GlutaMAX, and 0.5× B27 Supplement Minus Vitamin A. Finally, the cells were cultured for 11 days in hepatocyte culture medium (HCM, Lonza) without EGF but with 20 ng/mL oncostatin M (OsM; R&D Systems) and 3× GlutaMAX.
9
Directed Hepatic Differentiation of MSCs
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pPBHF3 and pPBase were co-transfected into MSCs with Lipofectamine 3000 (Invitrogen). pPBd and pPBase were transfected into MSCs in the same manner as mock samples (pPBd-TCs). After medium was changed, puromycin was added for antibiotic selection and when cells reached confluent the differentiation step was initiated.
For the first twelve days, as step A, cells were cultured in expansion Medium consisted of DMEM/F-12 (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin/streptomycin (Wako), 10 mM nicotinamide (Sigma Aldrich), 100 μM 2-melcaptomethanol (Wako), 100 μM ascorbic acid (Wako), 1 μM Dexamethasone (Dex) (Wako) and ITS (BD Biosciences), supplemented with 20 ng/ml hepatocyte growth factor (HGF) (R&D Systems) and 20 ng/ml epidermal growth factor (EGF) (R&D systems). Then step B was initiated with maturation medium consisted of IMEM + GlutaMax (Invitrogen) containing 0.5% bovine serum albumin (BSA) (Wako), 1% penicillin/streptomycin, 10 mM nicotinamide, 100 μM 2-melcaptomethanol, 500 μM ascorbic acid, 1 μM Dex and ITS, supplemented with 20 ng/ml oncostatin M (OSM) (R&D Systems) and 20 ng/ml EGF. For the first three days of the differentiation step, 200 uM 5′-azacytidine (5′-aza) was added to expansion medium.
For the first twelve days, as step A, cells were cultured in expansion Medium consisted of DMEM/F-12 (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin/streptomycin (Wako), 10 mM nicotinamide (Sigma Aldrich), 100 μM 2-melcaptomethanol (Wako), 100 μM ascorbic acid (Wako), 1 μM Dexamethasone (Dex) (Wako) and ITS (BD Biosciences), supplemented with 20 ng/ml hepatocyte growth factor (HGF) (R&D Systems) and 20 ng/ml epidermal growth factor (EGF) (R&D systems). Then step B was initiated with maturation medium consisted of IMEM + GlutaMax (Invitrogen) containing 0.5% bovine serum albumin (BSA) (Wako), 1% penicillin/streptomycin, 10 mM nicotinamide, 100 μM 2-melcaptomethanol, 500 μM ascorbic acid, 1 μM Dex and ITS, supplemented with 20 ng/ml oncostatin M (OSM) (R&D Systems) and 20 ng/ml EGF. For the first three days of the differentiation step, 200 uM 5′-azacytidine (5′-aza) was added to expansion medium.
10
Keratinocyte Cultivation and Treatment
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Normal human epidermal keratinocytes (NHEK) were obtained from surgical samples of healthy chest skin as previously described [39 (link)]. The use of these samples for research studies was approved by the Ethical Committee of the Poitiers Hospital (Poitiers, France). The cells were seeded in 96-well plates and cultured at 37 °C and 5% CO2 for 24 h in keratinocyte serum free medium supplemented with 0.25 ng/mL epidermal growth factor, 25 µg/mL pituitary extract, and 25 µg/mL gentamycin (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2. Then, they were treated or not (control) with the test compounds (LE and GAGs), alone or in combination, at 0.02 mg/mL each. In order to simulate stimulated conditions (skin inflammation), a cytokine mix containing oncostatin M (OSM; R&D systems, Minneapolis, MN, USA), interleukin 17 (IL-17; R&D systems, Minneapolis, MN, USA), and tumor necrosis factor α (TNF-α, R&D systems, Minneapolis, MN, USA), at 5 ng/mL each, was added to the medium (stimulated control). The cells were then incubated for 72 h at 37 °C and 5% CO2. All experimental conditions were performed in triplicate.
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