MECs were co-cultured with mitomycin C-treated C3H10T1/2 in DMEM/F12 supplemented with 10% FBS, 10 ng/mL human epidermal growth factor (hEGF; BD Biosciences, Tokyo, Japan), 5 μg/mL insulin (Wako), 0.5 μg/mL hydrocortisone (Sigma), 5 μM forskolin (Wako), 1.8 × 10−4 M adenine (Sigma), 100 μg/mL streptomycin (Meiji Seika Pharma), 100 U/mL penicillin G (Meiji Seika Pharma), 50 μg/mL gentamycin (Nakalai Tesque, Tokyo, Japan), 10 μM Rho-associated coiled-coil-forming kinase inhibitor (ROCKi; Y-27632; LC Laboratories, New Boston, MA, USA) [20 (link), 21 (link)], and 10% Matrigel (growth factor reduced; BD Biosciences) at 5% CO2 and 37 °C for 7 days. The numbers of MECs and C3H10T1/2 were 5000 and 6.25 × 104, respectively, in 250 μL of culture medium in a 48-well plate. When wells of different sizes were used, these numbers were changed proportionately relative to the area of the well.
Forskolin
Manufactured by Fujifilm
Sourced in Japan, United States
Forskolin is a laboratory equipment product manufactured by Fujifilm. It is used for various research and analysis purposes, providing a reliable and precise tool for scientific investigations. The core function of Forskolin is to facilitate the extraction and analysis of specific chemical compounds from various samples.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Insulin, by Fujifilm (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Pd0325901, by Fujifilm (3 mentions)
Chir99021, by Fujifilm (3 mentions)
Dexamethasone (dex), by Fujifilm (3 mentions)
Streptomycin, by Meiji Seika Pharma (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Sb431542, by Fujifilm (2 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (2 mentions)
Pifithrin α, by Fujifilm (2 mentions)
Ldn193189, by Fujifilm (2 mentions)
Thapsigargin, by Fujifilm (2 mentions)
Insulin, by Merck Group (2 mentions)
Retinoic acid, by Merck Group (2 mentions)
Recombinant human insulin, by Fujifilm (2 mentions)
L ascorbic acid 2 phosphate, by Cayman Chemical (2 mentions)
49 protocols using forskolin
1
Co-culture of MECs and C3H10T1/2 Cells
2
Forskolin-induced Neurodegeneration Model
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PC12, a rat pheochromocytoma cell line, differentiates in the presence of forskolin and is used as a model of neurodegeneration. PC12 cells were seeded in collagen-coated 24-well plates (Thermo Fisher Scientific Inc.) at a density of 5.0 × 103 cells/well (non-treated with chemotherapeutic agents) or 2.0 × 104 cells/well (treated with chemotherapeutic agents). Neurite outgrowth was induced by 10 µM forskolin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 24 h before exposure to drugs. The cells were treated with oxaliplatin (3 µM), cisplatin (10 µM), paclitaxel (3 nM), or bortezomib (300 nM) in the presence/absence of alogliptin (0.1–100 nM) or exendin-3 (9–39) amide (1 µM), the GLP-1 receptor antagonist, for 24 h. The concentrations of these drugs were determined based on previous reports30 (link). The living cells, which were not stained with trypan blue (Thermo Fisher Scientific Inc.), were analyzed using a phase-contrast microscope (CKX41; Olympus Co., Tokyo, Japan) after incubation. The neurite lengths were measured by ImageJ 1.51 software (Wayne Rasband, National Institutes of Health, MD, USA). Each experiment was performed in four wells per group.
3
Real-time Bioluminescence Monitoring of Cell Cultures
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Real-time bioluminescence analysis was performed according to the methods in previous
reports (Umemura et al., 2013 (link);
Umemura et al., 2014 (link)).
Briefly, the cells were seeded in 35-mm culture dishes or 24-well black plates, and the
medium was replaced with Prime ESC medium or the differentiating culture medium without
phenol red, comprising 0.2 mM luciferin (Promega, Madison, WI) and 15 mM HEPES. Cells were
treated with 100 nM dexamethasone (Sigma, St. Louis, MO) or 10 µM forskolin (Wako) for
synchronization. The dishes or plates were set on a turntable in an in-house fabricated
real-time monitoring system developed by Dr. Takao Kondo (Nagoya University, Nagoya,
Japan; Kiyohara et al., 2006 (link)).
The bioluminescence from each dish or well was measured for 1 min at 20-min intervals.
reports (Umemura et al., 2013 (link);
Umemura et al., 2014 (link)).
Briefly, the cells were seeded in 35-mm culture dishes or 24-well black plates, and the
medium was replaced with Prime ESC medium or the differentiating culture medium without
phenol red, comprising 0.2 mM luciferin (Promega, Madison, WI) and 15 mM HEPES. Cells were
treated with 100 nM dexamethasone (Sigma, St. Louis, MO) or 10 µM forskolin (Wako) for
synchronization. The dishes or plates were set on a turntable in an in-house fabricated
real-time monitoring system developed by Dr. Takao Kondo (Nagoya University, Nagoya,
Japan; Kiyohara et al., 2006 (link)).
The bioluminescence from each dish or well was measured for 1 min at 20-min intervals.
4
Organoid Generation from Primary Hepatocytes
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Three lots of PHHs (lots DOO; Celsis, HC10-10; XENOTECH, HC4-24; XENOTECH) were used to generate organoids (Table S1 ). PHHs were washed with cold Advanced DMEM/F12 (Thermo Fisher Scientific) and spun at 400 g for 5 min. The cell pellet was mixed with Matrigel (growth factor reduced, Corning) and 1 × 104 cells were seeded per well in a 24-well plate. After the Matrigel had solidified, 500 μl of organoid expansion medium was added to each well. The organoid expansion medium was prepared as described in a previous report [13 (link)]. Briefly, Advanced DMEM/F12 was supplemented with 1% Antibiotic Antimycotic Solution and 1 × GlutaMAX (GIBCO), 10 mM HEPES (Nacalai Tesque), 2% B27 supplement (GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma), 10 nM recombinant gastrin (Merk), 50 ng/ml EGF (R&D), 10% R-Spondin1 conditioned medium (homemade), 100 ng/ml recombinant human FGF10 (peprotech), 25 ng/ml recombinant human HGF (R&D), 5 μM A83-01 (Wako), and 10 μM Forskolin (Wako). During cultivation, the medium was refreshed every 3 days. For the establishment of the organoids, the medium was supplemented with 25 ng/ml Recombinant Noggin (R&D), 7.5 ng/mL Recombinant Wnt3a (R&D) and 10 μM Y27632 (Wako) for the first 3–4 days. Passage was performed in a 1:3 split ratio once every 10–14 days. Organoids passaged 5–10 times were used in all experiments.
5
Differentiation of iPSCs into Neural Stem Cells
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The HDDPC-derived iPSCs were cultured in NSC medium, based on N2B27 medium containing DMEM/F12 (#12634-010; Thermo Fisher Scientific, Inc., Waltham, MA, USA), Neurobasal medium (#21103-049; Thermo Fisher Scientific, Inc.), N2 supplement (#17502-048; Invitrogen, Carlsbad, CA, USA), and B27 supplement (#17504044; Invitrogen) in a ratio of 48:48:1:2, respectively. In the NSC medium, 5 ng/mL recombinant human bFGF, 0.02 μg/mL recombinant human LIF, 1 μM PD0325901 (#162-25291; Wako Pure Chemical Industries, Ltd.), 10 μM PD98059 (#169-19211; Wako Pure Chemical Industries, Ltd.), 3 μM CHIR99021 (#038-23101; Wako Pure Chemical Industries, Ltd.), 10 μM forskolin (#067-02191; Wako Pure Chemical Industries, Ltd.), and 1 μM kenpaullon (#110-00831; Wako Pure Chemical Industries, Ltd.) were also included. Medium change and cell passage were done as for cultivation of EpiSCs, as mentioned above.
6
Coumarin and Chrysin Bioactivity Assay
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6,7-Dimethoxycoumarin and chrysin were purchased from Sigma–Aldrich (St Louis, MO). Forskolin was obtained from Wako Pure Chemical (Osaka, Japan). Promoter vectors (pGL4.26, pGL4.50, and pGL4.74) and a Dual-Glo luciferase assay system were purchased from Promega (Madison, WI). All other chemicals and solvents were of analytical grade or the highest grade commercially available.
7
Induction of Fibroblast-Derived Neurons
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Four human primary fibroblasts were purchased via DS Pharma Medical (Osaka, Japan). Information on the fibroblasts is shown in Table 1 . Cells were seeded at 8 × 104 cells in a 35-mm dish in DMEM high-glucose medium (Gibco, Grand Island, NY) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. To generate induced neurons, on day 3, cells were switched from DMEM medium to neuronal medium containing DMEM/F12 [1% (vol/vol) N2 supplement, Gibco] and Neurobasal [2% (vol/vol) B27 supplement, Gibco] mixed at a 1:1 ratio. When indicated, small molecules were added to neuronal medium to reach the following final concentrations: SB-431542 (2 µM, Wako, Osaka, Japan), LDN-193189 (1 µM, Wako), CHIR99021 (1 µM, Wako), PD0325901 (1 µM, Wako), Pifithrin-α (5 µM, Wako) and Forskolin (7.5 µM, Wako).
8
Monitoring Intracellular cAMP in HEK293 Cells
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HEK293 cells were purchased from JCRB Cell Bank and cultured in E-MEM media with l -Glutamine and Phenol Red (Wako), containing 10% (vol/vol) fetal bovine serum (FBS) and penicillin–streptomycin. The cells were co-transfected with the plasmid carrying the Rh-PDE variant and the pGloSensor-22F cAMP vector (Promega), by using Lipofectamine 2000 (Invitrogen). Changes in the intracellular cAMP concentration of the HEK293 cells were measured by the GloSensor assay (Promega). The transfected cells were incubated with or without 0.5 μM all-trans-retinal (Toronto Research Chemicals). Before measurements, the culture medium was replaced with a CO2-independent medium, containing 10% (vol/vol) FBS and 2% (vol/vol) GloSensor cAMP stock solution (Promega). The cells were then incubated for 2 h at room temperature in the dark. The intracellular cAMP level was observed by monitoring the luminescence, using a microplate reader (Corona Electric) at 27 °C. The cells were treated with 3.5 μM forskolin (Wako), a direct activator of adenylyl cyclase, to elevate the intracellular cAMP level. The cells were illuminated with a xenon lamp (LAX-103, Asahi Spectra Co., Ltd., Japan) through an interference filter (510 nm). The light intensity was adjusted to 2.28 µW/mm2, and measured by an LP1 power meter (Sanwa Electric Instruments Co., Ltd., Japan).
9
Isolation and Culture of Human Biliary Epithelial Cells
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Normal human BECs were isolated, purified and cultured from human liver specimens, as described previously [32 (link)]. Human BECs were incubated with a culture medium composed of D-MEM/F-12, Nu-Serum (Becton Dickinson, Bedford, MA), ITS+ (Becton Dickinson), 5μM forskolin (Wako, Osaka, Japan), 12.5mg/ml of bovine pituitary extract (Gibco, Carlsbad, CA), 1μM dexamethasone (Sigma, St Louis, MO), 5μM Triiodo-thyronine (Sigma), 5mg/ml glucose (Sigma), 25mM sodium bicarbonate (Sigma), 1% antibiotics antimycotic, 20ng/ml of human epidermal growth factor (Gibco), and 10ng/ml human hepatocyte growth factor (Gibco). Total RNA prepared from the cultured BECs within 10 passages (n = 3) were used for quantitative RT-PCR analysis.
10
Antibodies and Reagents for SOCE Analysis
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Rabbit polyclonal antibody against ALG-2 was prepared as described previously [65 (link)]. The following antibodies were used for Western blotting: anti-STIM1 (ab57834) from Abcam (Cambridge, UK); anti-STIM2 from Alomone labs; anti-Orai1 (G-2, sc-377281), anti-GAPDH (6C5, sc-32233) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-NFAT1 (D43B1, #5861S) from CST Japan (Tokyo, Japan); HRP-conjugated goat antibodies against mouse IgG and rabbit IgG from Jackson Immunoresearch (West Gove, PA, USA). Reagents of biochemical or cell culture grade were purchased as follows: ionomycin from Cayman Chemical (Ann Arbor, MI, USA); BTP2 from Calbiochem/Merck Millipore (Temecula, CA, USA); carbachol (sc-202092) from Santa Cruz Biotechnology; forskolin, FK506, phorbol 12-myristate 13-acetate (PMA), and thapsigargin from Wako (Osaka, Japan); puromycin and blasticidin from InvivoGen (San Diego, CA, USA). A calcium assay Fura-2 kit was purchased from Dojindo (Kumamoto, Japan).
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