ADSCs (Lonza Group AG, Basel, Switzerland) were co-cultured with normal human dermal fibroblasts (NHDFs) as previously reported28 (link)30 (link). Briefly, NHDFs were seeded in six-well plates (IWAKI, Shizuoka, Japan) and cultured in Dulbecco's modified Eagle's Medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, while ADSCs were seeded on 0.4-µm Millicell® hanging cell culture inserts (Merck Millipore, Darmstadt, Germany) coated with type IV collagen (Nitta Gelatin, Osaka, Japan) and placed onto the plates. All-trans retinoic acid (Sigma-Aldrich) was added at 1 µM to the upper chamber. After culturing for 3 days, 25 ng/ml of bone morphogenetic protein 4 (R&D Systems, Minneapolis, MN, USA) was also added to the upper chamber. After 4 days, the media were replaced with keratinocyte serum-free medium (KSFM) (Thermo Fisher Scientific, Waltham, MA, USA). After 7 days of culture in KSFM, ADSCs were removed from the co-culture system and cultured on a dish coated with type IV collagen in KSFM for an additional 14 days.
Six well plates
Manufactured by Iwaki
Sourced in Japan
Six-well plates are a type of cell culture vessel used in laboratories. They provide six individual wells for culturing cells or performing assays. The plates are typically made of polystyrene, a common material for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Collagen type 4, by Nitta Gelatin (1 mentions)
Adscs, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
6 deep well plates, by Corning (1 mentions)
Aron alpha a tissue adhesive, by Daiichi Sankyo (1 mentions)
Mwco spectra por 4 dialysis membrane, by Spectrum Chemical (1 mentions)
Aron alpha a, by Daiichi Sankyo (1 mentions)
Streptomycin, by Fujifilm (1 mentions)
Lps from escherichia coli, by Merck Group (1 mentions)
Thp 1, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Thp 1, by Merck Group (1 mentions)
Penicillin g, by Nacalai Tesque (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
4 protocols using six well plates
1
Co-culture of ADSCs and NHDFs for Keratinocyte Differentiation
2
Dialysis-Culture System for Cell Studies
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The simple dialysis-culture system consists of upper culture compartment and lower dialysate compartment. A mesh bottom-cell strainer (PluriSelect, Leipzig, Germany) was modified by cutting and replacing the bottom mesh layer with a 12-kDa MWCO Spectra/Por 4 dialysis membrane (Spectrum Chemical, New Brunswick, NJ, USA). The dialysis membrane was affixed to the bottom of the strainer using an alkyl-α-cyanoacrylate-based surgical-grade tissue adhesive (Aron Alpha A; Daiichi Sankyo, Japan). The upper dialysis culture compartment inserts were then placed in 6-deep well plates (Corning, NY, USA). To represent the control condition, the cell strainer was directly affixed to the bottom surface of six-well-plates (Iwaki, Tokyo, Japan) using Aron Alpha A tissue adhesive (Daiichi Sankyo). All the devices were sterilized using an ethylene oxide gas sterilizer before use.
3
THP-1 Monocyte Differentiation and Activation
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The human monocytic cell line, THP-1 (JCRB0112.1; JCRB Cell Bank, Osaka, Japan), was cultured in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA), supplemented with 5% heat-inactivated fetal bovine serum (FBS; CORNING, NY, USA), penicillin G (100 U/ml) (Nacalai Tesque, Kyoto, Japan), and streptomycin (100 mg/ml; Wako Pure Chemical Industries, Osaka, Japan) at 37 °C with 5% CO2. THP-1 cells were seeded at 2 × 106 cells/well in six-well plates (Iwaki, Chiba, Japan) and cultured in RPMI 1640 medium containing 5% FBS and 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA). After culturing overnight, cells were washed with phosphate-buffered saline (PBS; pH 7.2). THP-1 cells were then treated with or without Zol (10 μM; Sigma-Aldrich). After culturing for an additional 24 h with or without Zol, LPS from Escherichia coli (100 ng/ml; Sigma–Aldrich) or IL-4 (50 ng/ml; R&D Systems, Minneapolis, MN, USA) were added.
4
Classifying Cell Locomotion Dynamics
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Cells were seeded onto glass bottomed Iwaki six-well plates (Iwaki, Japan) and then treated with DEX 10 -7 M, TGF-β2 or decorin 25 µg/ml for 7 days after which the plates were placed into a microscope holder inside an incubator that controlled the temperature (37°C) and live phase contrast images were recorded over 20 hour period using a Zeiss microscope with a X 20 objective. At the end of the phase contrast filming period the cells were fixed in 10% NBF and stained as above. After staining the cells were analysed on the basis of clear fluorescent staining and their appearance was easy to classify. We then looked at the three classifications of cell shape and followed their life history in the culture. An arbitrary scale for locomotion of the cells was employed where M* cells moved at 4µm/hour or less, M** reached up to 12µm/hour, whereas above that M*** cells were faster still and some could reach a movement rate of 25µm/hour for short periods, M**** is for cells that moved extremely quickly in the culture. Speed was calculated by distance and time.
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