Suspended NPCs (NS1) were used for MN induction as described previously [28 ], with minor modifications. In short, mature neurospheres (>300 μM) were treated with TrypLE Select to make single-cell suspensions, count the cell number, and start adherent culturing in KBM with micronutrient-specific supplements. The supplements included 2% vitamin B27, 1% N2 (Gibco, cat. 17,502–048), 10 ng/ml recombinant human brain-derived neurotrophic factor (BDNF) (R&D system, cat. 248-BD), 10 ng/ml recombinant human glial cell line-derived neurotrophic factor (GDNF) (R&D system, cat. 212-GD), 50 ng/ml small molecule recombinant human Sonic Hedgehog (hSHH) (R&D system, cat. 1845-SH), 200 ng/ml ascorbic acid (Wako chemicals, cat. 012–04802), 1 mM dibutyl cyclic AMP (Sigma–Aldrich, cat. D0627), 10 ng/ml recombinant human insulin-like growth factor-1 (R&D system, cat. 291-G1), and 50 nM retinoic acid (Wako chemicals, cat, 182–01116). The cells were seeded on poly-L-ornithine (Sigma–Aldrich, cat. P3655) and laminin (Invitrogen, cat. 23,017–015)-coated cell desk LF1 (Sumilon, cat, MS92132) or culture plates. The culture medium was changed with the same medium on alternate days. When the color of the medium was altered in one day, the medium was changed every day until 4 weeks (28 days) of culture.
Retinoic acid
Manufactured by Fujifilm
Sourced in Japan
Retinoic acid is a laboratory compound used in various research applications. It is a metabolite of vitamin A and plays a role in cellular differentiation and growth. Retinoic acid is commonly used in cell culture studies and biomedical research, but its specific function and intended uses should not be extrapolated beyond a factual description of its core properties.
Automatically generated - may contain errors
Lab products found in correlation
Y 27632, by Fujifilm (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
Dibutyl cyclic amp, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Ril 2, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Mirna mimic control, by Horizon Discovery (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions)
Purmorphamine, by Cayman Chemical (1 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
7 protocols using retinoic acid
1
Motoneuron Induction from Neural Stem Cells
2
Murine MLN Cell Differentiation
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The prepared MLNs cells from R23-3 or RD10 mice were dispensed into 96-well plates at
2.5×105 cells/well and incubated for 72 hr in 5% fetal calf serum (FCS)-RPMI
medium in a 5% CO2 incubator at 37°C in the presence of the following materials
at their final concentrations: OVA (0.25 mg/mL, Sigma-Aldrich), TGF-β (2 ng/mL, R&D
Systems, Minneapolis, MN, USA), retinoic acid (1 μM, FUJIFILM Wako Pure Chemical Corp.),
and rIL-2 (2 µg/mL, eBioscience, Waltham, MA, USA). After culturing, the collected cells
were used for analysis by flow cytometry to analyze Foxp3 expression and the supernatant
was collected to analyze IL-4 production.
2.5×105 cells/well and incubated for 72 hr in 5% fetal calf serum (FCS)-RPMI
medium in a 5% CO2 incubator at 37°C in the presence of the following materials
at their final concentrations: OVA (0.25 mg/mL, Sigma-Aldrich), TGF-β (2 ng/mL, R&D
Systems, Minneapolis, MN, USA), retinoic acid (1 μM, FUJIFILM Wako Pure Chemical Corp.),
and rIL-2 (2 µg/mL, eBioscience, Waltham, MA, USA). After culturing, the collected cells
were used for analysis by flow cytometry to analyze Foxp3 expression and the supernatant
was collected to analyze IL-4 production.
3
Functional Validation of miRNA-664a-5p
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The miRNA-664a-5p mimic and miRNA mimic control were purchased from Dharmacon (Lafayette, CO, USA). The cells were transfected with 10 nM miRNA-664a-5p mimic or 10 nM pre-miR-664a using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 24 h at 37 °C and 5% CO2. For control experiments, the cells were also transfected with miRNA mimic control or pre-miRNA-U1A control. The next day, the medium was replaced with fresh growth medium or differentiation medium containing 30 µM retinoic acid (Wako Pure Chemical Industries, Osaka, Japan). The cells were again transfected with miRNA mimics, pre-miR-664a, or pre-miRNA-U1A control in growth medium or differentiation medium after 2 days. After 24 h, the medium was replaced with fresh growth medium or differentiation medium, and the cells were cultured for an additional 2 days.
To induce apoptosis, the cells were transfected with 10 nM pre-miR-664a or pre-miR-664-U1A using Lipofectamine 3000 (Invitrogen) for 24 h at 37 °C and 5% CO2. For control experiments, the cells were also transfected with pre-miRNA-U1A control.
To induce apoptosis, the cells were transfected with 10 nM pre-miR-664a or pre-miR-664-U1A using Lipofectamine 3000 (Invitrogen) for 24 h at 37 °C and 5% CO2. For control experiments, the cells were also transfected with pre-miRNA-U1A control.
4
Osteogenic Induction of hiPSCs Using Collagen Gel
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The osteogenic induction of hiPSCs using a previously reported method17 (link) was performed on a dish or plate coated with porcine type I collagen gel (Cellmatrix, Nitta gelatin). In brief, 1.5 X 106 or 6 X 105 hiPSCs were seeded in a 3.5-cm glass bottom dish (for confocal imaging) or a 12-well plate (for RNA extraction and tissue sectioning) coated by collagen gel and cultured with a mixed medium of mTeSR1 (20%) and osteogenic induction (OI) medium (80%), which was composed of KnockOut™ DMEM (Thermo) supplemented with 20% FBS (Nichirei), 2 mM Gluta-MAX ™ (Thermo), 10 mM glycerol-2-phosphate (Sigma), 1 nM dexamethasone (Sigma), 0.1 mM 2-mercaptoethanol (Thermo), 50 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Nacalai Tesque), and 1% non-essential amino acids (Thermo) in the presence of Y-27632 (10 μM, Wako) and 1 μM retinoic acid (Wako). On day 2, the mixed medium was replaced with 100% OI medium and refreshed at days 4, 7, 9, and 11.
5
Cell Culture Conditions for Various Cell Lines
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HEK293T and HeLaS3 cells were cultured in DMEM (Sigma) containing 10% FBS (GE Healthcare), nonessential amino acids (NEAA) (Gibco) and 1% penicillin–streptomycin (Wako). SH-SY5Y cells were maintained in DMEM/F12 (Sigma) containing 10% FBS, 1% penicillin–streptomycin and NEAA. hiPSCs (201B7) were purchased from RIKEN Cell Bank and maintained in StemFit medium (Ajinomoto). Neurospheres were produced and maintained according to a previous report56 (link). Briefly, cells were cultured in KBM neural stem cell medium (Kohjin Bio) supplemented with 2% B27 (Gibco), 20 ng/mL bFGF (Wako), 10 ng/mL LIF (Millipore), 10 ng/mL Y-27632 (Wako), 3 μM CHIR (Cayman Chemical Company), 2 μM SB431542 (Sigma), 1 μM retinoic acid (Wako), and 1 μM purmorphamine (Cayman Chemical Company).
6
Culturing Caco-2 and SH-SY5Y Cell Lines
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The human colorectal cancer cell line Caco-2 (ATCC, Manassas, VA, USA) and the human neuroblastoma cell line SH-SY5Y (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Gaithersburg, MD, USA) at 37 °C in an atmosphere containing 5% CO2. γ-Aminobutyric acid (GABA) was purchased from Abcam (Cambridge, UK). 5-Aminoimidazole-4-carboxamide 1-β-d -ribofuranoside (AICAR) and retinoic acid (RA) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan).
7
Myotube Differentiation of Human iPSCs
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Myotube differentiation of human iPS cells was performed as previously described [8 (link),11 (link)]. On day 0, N-iPS cells, D-iPS and R-iPS cells were seeded at densities of 3.0 × 105, 5.0 × 105, and 1.0 × 105 cells/well, respectively, onto 6-well plates coated with Matrigel (354230; Corning, New York, NY, USA). Since the growth rate differed depending on the type of iPS cells, the seeding cell density was changed so that cells reach semi-confluency during the same period. Cells were cultured in StemFit AK02N medium supplemented with 10 µM Y-27632, 0.1, 1.0 or 10 µM retinoic acid (RA) (Fujifilm Wako Pure Chemical, Osaka, Japan), and 0.5 µg/mL puromycin or 0.1 mg/mL neomycin. The next day, cells were cultured in ReproStem medium (ReproCELL, Yokohama, Japan) supplemented with the corresponding concentrations of RA and antibiotics. From day 2, doxycycline (Dox) (Sigma-Aldrich) was added to the medium at a concentration of 1.0 µg/mL. On day 3, the culture medium was changed to α-MEM (Invitrogen, Carlsbad, CA, USA) containing 5% knock-out serum replacement (Invitrogen), supplemented with Dox and RA. Cells were cultured for 14 days and the medium was changed with fresh medium every day.
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