We used the MTT assay to check cell viability. We seeded the sebocytes and ORS cells in 96-well collagen-coated plates at a density of 5,000 cells/well (Becton Dickinson, Franklin, NJ, USA) for 24 h. We added various concentrations (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, and 100 µg/ml) of PM10 (Sigma-Aldrich, St. Louis, MO, USA) and various concentrations (0.5 mg/ml, 1 mg/ml, 10 mg/ml, 30 mg/ml, and 50 mg/ml) of SHE to the well plates of the sebocytes and ORS cells for 1 day or 3 days each and the MTT solution (3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide) at 70 µg/well for 3 h. We solubilized the formazan produced with dimethyl sulfoxide (DMSO) and measured the optical density at 570 nm.
Collagen coated plates
Manufactured by BD
Sourced in United States
Collagen-coated plates are laboratory equipment designed to facilitate cell culture experiments. The plates are pre-coated with collagen, a naturally occurring extracellular matrix protein. This coating provides a substrate that promotes cell adhesion and growth, supporting various cell-based studies.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12 medium, by Merck Group (2 mentions)
Gene pulser xcell total system, by Bio-Rad (2 mentions)
Transit mrna transfection kit, by Mirus Bio (2 mentions)
Hepg2 cells, by European Collection of Cell Cultures (2 mentions)
Dcf da, by BD (1 mentions)
Fluorescence microplate reader, by Molecular Devices (1 mentions)
Dieckol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by American Type Culture Collection (1 mentions)
Supporting culture medium, by Xenotech (1 mentions)
Plc prf 5, by Korean Cell Line Bank (1 mentions)
Hep3b, by Korean Cell Line Bank (1 mentions)
Fa2n 4, by Xenotech (1 mentions)
Hep3b, by Welgene (1 mentions)
Plc prf 5, by Welgene (1 mentions)
Snu449, by Welgene (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Welgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Nucleofection protocol, by Lonza (1 mentions)
13 protocols using collagen coated plates
1
Evaluating Cytotoxicity of PM10 and SHE on Sebocytes and ORS Cells
2
Measuring ROS Production in Sebocytes and Keratinocytes
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ROS production in sebocytes and ORS keratinocytes was assessed by measuring 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, Invitrogen, Carlsbad, CA, USA). The sebocytes and ORS keratinocytes were seeded onto six-well collagen-coated plates (Becton Dickinson) at 5 × 105 cells/well for 24 h. Pre-labeled cells with 10 μM DCF-DA were washed with PBS for 30 min, and then were treated with 100 μg/mL of PM10 and 5 μM punicalagin, 5 μM dieckol, 1 μM EGCG, 1 μM resveratrol, or 10 μg/mL SHE, and incubated at 37 °C for 2 h in the dark. Cells were extracted with 20 mM Tris-Cl buffer containing 1% sodium dodecyl sulfate (SDS) and 2.5 mM ethylene-diamine-tetraacetic acid (EDTA). After centrifuging at 13,000 rpm for 15 min, supernatants were measured using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) at excitation at 485 nm and emission at 538 nm.
3
Evaluating Sebocyte and ORS Cell Viability
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We used the MTT assay to check cell viability. We seeded the sebocytes and ORS cells in 96-well collagen-coated plates at a density of 5,000 cells/well (Becton Dickinson, Franklin, NJ, USA) for 24 h. We added various concentrations of dieckol (Sigma) to the well plates of the sebocytes and ORS cells for 3 days and the MTT solution (3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide) at 70 µg/well for 3 h. We solubilized the formazan produced with dimethyl sulfoxide (DMSO) and measured the optical density at 570 nm.
4
Isolation and Culture of Primary Human Hepatocytes
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Once the cells (mpPHH) were isolated and purified with two rounds of Percoll, they were freshly seeded on collagen-coated plates (BD Biosciences) in W10 plating medium. The cells were equally distributed by shaking on a flat surface and then, left on the bench at room temperature for 45 min. Once the cells settled and evenly distributed, they were transferred to a humidified 37 °C incubator. Cryopreserved PHHs were first thawed at 37 °C and then, transferred to 50 mL W10. After centrifugation at 50 × g, the cells were resuspended in W10, counted, and seeded on plates as described for mpPHH. For each plating format, we optimized the seeding density to achieve confluent cultures. To avoid concentrating cells in the middle of the well, we seeded the cells with excess medium. The next day, the cells were washed once with WEM to remove any cell debris and serum. For maintenance medium, we used hepatocyte-defined medium (HDM; catalog no. 05449; Corning) supplemented with 1% penicillin/streptomycin, 1% 200 mM l -glutamine, 0.1% 50 mg/mL Gentamicin, and 2% DMSO (catalog no. 4-x-5; ATCC). Unless otherwise stated, medium was changed every other day. Table 1 summarizes the different formats with seeding densities and medium volumes.
5
Establishment and Maintenance of HCC Cell Lines
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The Hep3B, Huh7, PLC/PRF/5, and SNU449 HCC cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). Huh7.5 cells were kindly provided by Dr. Marc Windisch (Institut Pasteur Korea, Gyeonggi-do, Korea), and Huh6 cells were obtained from Cell Bank Australia (Westmead, NSW, Australia). Human immortalized hepatocytes (Fa2N-4) were obtained from XenoTech (Lenexa, KS, USA). All cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Dulbecco’s Modified Eagle’s Medium (Welgene, Daegu, Korea) was used to cultivate the Hep3B, Huh7.5, and Huh6 HCC cell lines, and Roswell Park Memorial Institute 1640 (RPMI1640) medium was used to cultivate the Huh7, PLC/PRF/5, and SNU449 HCC cell lines. DMEM and RPMI1640 media were supplemented with heat inactivated 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1× penicillin-streptomycin (P/S; Gibco). Fa2N-4 cells were plated on collagen-coated plates (BD Biosciences, San Jose, CA, USA) in serum-containing plating medium (plating media; XenoTech), which was replaced with supporting culture medium (XenoTech) after cell attachment (approximately 3–6 h).
6
Hepatocyte Isolation and Culture Protocol
7
Isolation of Stromal Vascular Fraction
8
Transfection of HepG2 and PHH cells
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HepG2 cells and PHHs were transfected for 24 or 48 h with corresponding constructs; HepG2 cells were transfected using Lipofectamine/LTX and Opti-MEM with standard protocols (Life Technologies), while PHHs were transfected using a nucleofection protocol optimized for primary cells (Lonza). Briefly, for each transfection, 2×106 fresh PHHs were resuspended in 100 μL of optimized nucleofection buffer L3 and transfected with 5 μg of corresponding constructs using 4D-nucleofector (Lonza). Immediately after transfection, dead cells were separated by centrifugation (4 min, 570 rpm) of the transfection mix overlaid with 900 μL of Ficoll (GE Healthcare) and 750 μL of supernatant with dead cells was removed. The remaining cells were resuspended in InVitroGRO CP media with Torpedo antibiotic mix (Bioreclamation IVT), plated onto collagen-coated plates (BD Biosciences), and incubated overnight. Unattached and dead cells were removed 12 h post-transfection by replacing the transfection media with InVitroGRO HI culture media with Torpedo antibiotic mix (Bioreclamation IVT).
9
Huh-7.5 Cell-based HCV Transfection Assay
10
Huh-7.5 Cell-based HCV Transfection Assay
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At 24 h before transfection, 7.5×104 Huh-7.5 cells were seeded onto a 24-well plate. One day later, media were replaced with fresh media, and the cells transfected with 0.25 μg (per well) HCV RNA encoding GLuc using the TransIT mRNA transfection kit (Mirus) according to the manufacturer’s protocol. After 6 h incubation at 37°C, supernatant fluids were removed for GLuc assay and replaced with fresh media containing compound. Alternatively, 10 μg of HCV RNA was mixed with 5×106 Huh-7.5 cells and electroporated into cells using a Gene Pulser Xcell Total System (Bio-Rad) as described previously52 (link). Transfection of wild-type HCV RNA was performed by electroporating 5 μg HCV RNA in 2.5×106 Huh-7.5 cells and seeded into collagen-coated plates (BD Biosciences). Cells were grown in DMEM supplemented with 25 mM HEPES, 7 ng ml−1 glucagon, 100 nM hydrocortisone, 5 μg ml−1 insulin, 2 mM GlutaMAX, antibiotics, and 2% FBS. Culture supernatants were replaced with the media supplemented with drugs at 6 h and every 48 h thereafter and assayed for GLuc activity.
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