To establish primary fibroblast cultures, skin biopsies (1–2 mm in diameter) were taken from ears of CC004 and CC041mice. Biopsies were digested in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, Logan, UT) containing 2.5 mg/ml collagenase D (Gibco, Gaithersburg, MD) and 1.25 mg/ml pronase (Millipore, Burlington, MA) for 90 mins to isolate fibroblasts. Bmal1-dluc reporter gene (Addgene, Cambridge, MA) was delivered to fibroblasts by lentiviral-mediated gene delivery (VectorBuilder, Chicago, IL). Fibroblast rhythms were measured four days following viral transfection by synchronizing with 15 μM forskolin (Sigma, St. Louis, MO) for 2 hrs, then placed into DMEM recording media (15 μM forskolin, 25 mM HEPES (Gibco), 292 μg/ml L-glutamine (HyClone), 100 units/ml penicillin (HyClone), 100 μg/ml streptomycin (HyClone) and 10 μM luciferin (Promega, Madison, WI)) for bioluminescence analysis. Bioluminescence rhythms of Bmal1-dLuc were analyzed by an automated 32-channel luminometer (LumiCycle, ActiMetrics, Wilmette, IL) in a standard tissue culture incubator at 32°C. Bioluminescence rhythms were continuously recorded for ~70 secs at intervals of 10 mins over 7 days. The period and amplitude of molecular rhythms were determined from baseline-subtracted data using the damped sine fit and Levenberg-Marquardt algorithm (Izumo et al. 2003 (link)).
Forskolin
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Forskolin is a laboratory reagent used in scientific research. It is a chemical compound extracted from the roots of the Coleus forskohlii plant. Forskolin is commonly used as a tool compound in cell biology and biochemistry studies to investigate the function and regulation of the enzyme adenylate cyclase.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Acquire and analyze software, by Physiologic Instruments (2 mentions)
Vcc mc8, by Physiologic Instruments (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Forskolin, by Cell Signaling Technology (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (2 mentions)
5977b mass, by Agilent Technologies (2 mentions)
Geranylgeraniol, by Santa Cruz Biotechnology (2 mentions)
Vf 5ht, by Agilent Technologies (2 mentions)
Dpcpx, by Bio-Techne (2 mentions)
Picrotoxin, by Bio-Techne (2 mentions)
Cgp 52432, by Bio-Techne (2 mentions)
Platelet derived growth factor, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Thermo Fisher Scientific (1 mentions)
29 protocols using forskolin
1
Establishing Fibroblast Cultures for Circadian Analysis
2
Differentiation of Cells into Neurons
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Neuronal differentiation was induced by culturing cells at 90% confluence in DMEM/F-12 (1 : 1) medium supplemented with 2% (v/v) FBS, 100 ng/mL bFGF, 10 μM forskolin (Sigma) for 2 days, followed by culturing in DMEM/F-12 (1 : 1) medium supplemented with 2% FBS, 50 ng/mL BDNF (PeproTech), 50 ng/mL nerve growth factor (NGF; R&D Systems, Wiesbaden, Germany), 10 ng/mL NT3 (PeproTech), 5 μM forskolin, and 2% (v/v) B-27 (Gibco) for 5 days. The differentiated cells were evaluated with immunofluorescence staining for the expression of neuron-specific β3-Tubulin. For quantification, images of 6 fields per well were randomly selected by fluorescence microscopy at 200× magnification and the differentiation efficiency was expressed as the ratio of β3-Tubulin-positive cells with neuron-like morphology to the total number of cells labeled with Hoechst 33342.
3
Forskolin Extraction and Cell Assay
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forskolin, from Coleus forskohlii, ≥98% (HPLC) was purchased from Merck (Darmstadt, Germany). A 100 mM stock solution in dimethylsulfoxyde (DMSO, PanReac, Barcelona, Spain) was prepared and stored at −20 °C. Dilutions in culture medium were prepared just before the cell incubation. The same final concentration of DMSO on cells was used for all concentrations of forskolin, i.e., 0.1%.
Cell culture reagents: MEM medium, phosphate buffered saline (PBS), trypsin, fetal bovine serum (FBS), penicillin-streptomycin and glutamine were purchased from Gibco Life Technologies (Paisley, UK). Flasks and 96-well microplates were purchased from Corning (Amsterdam, The Nederlands).
A YO-PRO-1 (cat. no. Y3603) fluorescent probe was obtained from ThermoFisher Scientific.
Fluorescence Resonance Energy Transfer (FRET, cat. no. 62ESTPEG and 6FPROPEG) and ELISA (cat. no. MBS705577 and MBS764095) kits were purchased from Cisbio Biosassays (Codolet, France) and MyBioSource (Vancouver, BC, Canada), respectively.
Cell culture reagents: MEM medium, phosphate buffered saline (PBS), trypsin, fetal bovine serum (FBS), penicillin-streptomycin and glutamine were purchased from Gibco Life Technologies (Paisley, UK). Flasks and 96-well microplates were purchased from Corning (Amsterdam, The Nederlands).
A YO-PRO-1 (cat. no. Y3603) fluorescent probe was obtained from ThermoFisher Scientific.
Fluorescence Resonance Energy Transfer (FRET, cat. no. 62ESTPEG and 6FPROPEG) and ELISA (cat. no. MBS705577 and MBS764095) kits were purchased from Cisbio Biosassays (Codolet, France) and MyBioSource (Vancouver, BC, Canada), respectively.
4
Isolation and Culture of Rat SKP-SCs
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SKP-SCs isolated from the back skin of newborn green fluorescent protein–transgenic Sprague-Dawley rats were supplied by Nantong University (Wang et al., 2016; Zhu et al., 2018; Chen et al., 2020; Ma et al., 2021; Yan et al., 2022). The cells were cultured in Dulbecco’s Modified Eagle Medium/F12 (3:1, Gibco, Billings, MT, USA) containing 1% fetal bovine serum (Gibco), 2% N2 (Gibco), 5 µM forskolin (Gibco), 50 ng/mL heregulin-1β (Gibco), and 1% penicillin/streptomycin (Beyotime, Shanghai, China). High-purity cultures were obtained by serial passage. Cells at passages 18–20 were used in this study.
SKP-SCs were cultured at a concentration of 4 × 105 cells/cm2 for 48 hours, after which the conditioned medium was collected by centrifugation at low speed (4000 × g) for 10–15 minutes and filtered using a 0.22-μm syringe filter. The filtered SKP-SCs-CM was then concentrated by ultrafiltration (Amicon Ultra-15, Millipore, Billerica, MA, USA) and stored at –80°C until use.
SKP-SCs were cultured at a concentration of 4 × 105 cells/cm2 for 48 hours, after which the conditioned medium was collected by centrifugation at low speed (4000 × g) for 10–15 minutes and filtered using a 0.22-μm syringe filter. The filtered SKP-SCs-CM was then concentrated by ultrafiltration (Amicon Ultra-15, Millipore, Billerica, MA, USA) and stored at –80°C until use.
5
BRET Assay for CB1R and CB2R
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HEK-CRE cells were transfected with CB1R-GFP2 or CB2R-GFP2. Forty-eight hours post-transfection cells were washed twice with cold PBS and suspended in BRET buffer. Cells were dispensed into 96-well plates (10,000 cells/well) and treated with 10 μM forskolin and ligands (PerkinElmer). Media was aspirated from cells and cells were lysed with passive lysis buffer for 20 min at room temperature (Promega, Oakville, ON, Canada). Twenty microliters of cell lysate were mixed with luciferase assay reagent (50 μM; Promega, Oakville, ON, Canada) and light emissions were measured at 405 nm using a Luminoskan Ascent plate reader (Thermo Scientific, Waltham, MA, United States), with an integration time of 10 s and a photomultiplier tube voltage of 1200 V. Data are presented as % inhibition of forskolin response.
6
Circadian Regulation of Nampt Rhythms
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Sodium palmitate and FK866 were purchased from MilliporeSigma. Sodium palmitate was dissolved in molecular grade water by heating to 70°C to obtain a concentration of 50 mM. FK866 was dissolved in dimethyl sulfoxide (DMSO) (Thermo Scientific) to 10 μM. Treatments were diluted 1:1,000 in low glucose DMEM to reach the final concentrations as indicated in the results section. Cells were grown to to 75–80% confluency on 60 mm plates prior to treatment.
For time course experiments, mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F were grown to 75–80% confluency on 60 mm plates, serum-starved for 12 h and synchronized for 30 min with 20 μM forskolin (Thermo Scientific) in DMEM containing 5% FBS. This was then replaced with fresh DMEM containing 5% FBS, indicating t = 0 h. Cells were first harvested at 6 h, then afterwards at every 6-h interval up to 36 h. The samples used to assess Nampt rhythmicity were the same as those used in the study by Clemenzi et al. (31 (link)). With these studies, circadian oscillations of the core clock genes were demonstrated in the mHypoA-BMAL1-WT/F cells, and these rhythms were completely absent in the mHypoA-BMAL1-KO/F cell line.
For time course experiments, mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F were grown to 75–80% confluency on 60 mm plates, serum-starved for 12 h and synchronized for 30 min with 20 μM forskolin (Thermo Scientific) in DMEM containing 5% FBS. This was then replaced with fresh DMEM containing 5% FBS, indicating t = 0 h. Cells were first harvested at 6 h, then afterwards at every 6-h interval up to 36 h. The samples used to assess Nampt rhythmicity were the same as those used in the study by Clemenzi et al. (31 (link)). With these studies, circadian oscillations of the core clock genes were demonstrated in the mHypoA-BMAL1-WT/F cells, and these rhythms were completely absent in the mHypoA-BMAL1-KO/F cell line.
7
Comprehensive Oxidative Stress Assay Protocol
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Amplex Red and AbGreen-indicator H2O2 detection reagents were purchased from Thermo Fisher Scientific (A12222) (Waltham, MA) and Abcam (ab138874) (Waltham, MA), respectively. DiaAlk (PubChem CID: 134688955) (1-(tert-Butyl) 2-(2-methyl-4-(prop-2-yn-1-yloxy) butan-2-yl) (E)-diazene-1,2-dicarboxylate, 8 mM in DMSO) was acquired from Aobious Inc (Gloucester, MA), biotin-azide (3aS,4 S,6aR)-N-(3-azidopropyl) hexahydro-2-oxo-1 H-thieno[3,4-d] imidazole-4-pentanamide, 5 mM in DMSO) and VAS2870 (PubChem CID: 4058452) were purchased from Cayman Chemicals (Ann Arbor, MI), (−)-isoproterenol bitartrate (ISO, PubChem CID:160420) (I2760), salbutamol (SAL, PubChem CID: 2083) (S8260), and N-Acetyl-L-cysteine (NAC, PubChem CID:12035) (A7250) were purchased from Millipore Sigma (St. Louis, MO); 3-isobutyl-1-methylxanthanine (IBMX, PubChem CID:3758) (PHZ1124), forskolin (FSK, PubChem CID:47936) (BP252010), dithiothreitol (DTT, PubChem CID:446094), and 4,4′-dithiodipyridine (4,4-DPS, PubChem CID: 75846) (162240050) were purchased from Thermo Fisher (Carlsbad, CA). ICI-118,551 hydrochloride (PubChem CID: 11957590) (0821) was from Tocris Bioscience (Bristol, UK). All other reagents were purchased at their highest available purity from Millipore Sigma or Fisher Scientific.
8
Purification and Propagation of Schwann Cells
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Schwann cell cultures at passage 11 were taken from storage in liquid nitrogen and thawed as required. These cells were previously derived from primary neonatal Schwann cells (SCs) obtained from Wistar RjHan:WI rat pups and cultured and purified following a previously published protocol [48 (link)] and in accordance to [30 (link)]. In brief, sciatic nerves were collected and enzymatically digested. The separated cells were cultured in poly-l-lysine (PLL)-coated dishes containing Dulbecco’s modified Eagle’s medium supplemented with 0.1 µM Forskolin, 1% Pen/strep, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum (FCS) (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2 in a humidified atmosphere. PLL coating was performed by covering the culture dishes with PLL for 45 min at room temperature. After removing the PLL, plates were washed 2 times with Ampuwa® (Fresenius Kabi, Bad Homburg vor Höhe, Germany). To prevent excessive fibroblast contamination, 1 mM of arabinoside-c (Sigma-Aldrich, St. Louis, MO, USA) was added at 2 and 3 days in vitro, respectively. The cells were finally purified to >90% by immunopanning, purity was controlled in immunocytochemistry, and cells propagated in medium as described above, just with an increased concentration of 2 µM Forskolin [30 (link)].
9
Intracellular cAMP Measurement in HUVECs and HaCaT Cells
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The measurement of intracellular cAMP levels was performed using the cAMP-GloTM Assay (PromegaTM Corporation, Madison, WI, USA). HUVECs and HaCaT cells (2500 cells/well) were seeded on tissue culture-treated Nunc white MicroWell 96-Well Optical-Bottom Plates (Thermo Fisher Scientific Inc., Waltham, MA, USA) and grown overnight. Cells were exposed to odorants, as detailed above, in serum-free media for 30 min, with a 10 μM forskolin (Thermo Fisher Scientific Inc.) exposure as the positive control. The cells were lysed, and luminescence was analyzed using a microplate-reader (Synergy™ HT, BioTek, Winooski, VT, USA) according to the manufacturer’s protocol. The cAMP activity was represented as cAMP activity relative to controls within each experiment.
10
Differentiation of Schwann Cells from Precursors
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For differentiation into SCs, SCPs were cultured on Matrigel-coated plates in SCDM containing 1% FBS, 200 ng/mL NRG1, 4 μM forskolin (Sigma), 100 nM all-trans RA (Sigma) and 10 ng/mL PDGF-BB in DMEM/low glucose. After 3 days of incubation, the culture medium was replaced with SCDM containing 1% FBS, 200 ng/mL NRG1, and 10 ng/mL PDGF-BB (Thermo Fisher Scientific), but not forskolin or RA. After another 2 days of incubation, the culture medium was replaced with SCDM containing 1% FBS and 200 ng/mL NRG1 but not forskolin, RA, or PDGF-BB (Schwann cell medium [SCM]). The cells were maintained in SCM for expansion. SCs were generated after approximately 2–3 days of incubation in SCM.
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