Cells growing in monolayers in Matrigel-coated cell culture flasks cultured in NESC culture medium, as previously described, were detached with pre-warmed StemPro Accutase Cell Dissociation Reagent for 3–5 min at 37ºC. Then, cells were diluted in KnockOut DMEM (Gibco) and precipitated through centrifugation at 1100 RPM for 5 min. For cell differentiation in neural cultures composed of neurons and glia, 250 000 cells/well (MW12 plates) were plated on Matrigel-coated coverslips in N2B27 medium supplemented with 250 μM Dibutyryl cyclic-AMP sodium salt (dbcAMP) (BIOLOG), 5 μM Forskolin (TargetMol), and 2 μM retinoic acid (Sigma) as previously reported5 ,46 (link). Cells were maintained for up to 2 months and the culture medium was changed every five days.
Forskolin
Manufactured by Targetmol
Sourced in United States
Forskolin is a compound extracted from the root of the Coleus forskohlii plant. It is a natural polypeptide that acts as an activator of the enzyme adenylate cyclase, which plays a role in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Ta2 fl, by Nikon (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Sb431542, by Targetmol (1 mentions)
Pyrrolidinedithiocarbamic acid pdtc, by Beyotime (1 mentions)
Sq22536, by Abcam (1 mentions)
3 protocols using forskolin
1
Differentiation of Neural Cells in Vitro
2
Hepatic Differentiation and Maturation Protocol
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For monolayer hepatic-differentiation, iHepLPCs were seeded and kept in TEM for 2-4 days until they reached full confluence. The medium was then changed to modified Hepatocyte Maturation Medium (mHMM) 12 (link), 13 (link), 16 (link) and cultured for 9-15 days for further maturation; medium was changed every day. The modified HMM (mHMM) comprised of DMEM/F12 supplemented with N2 and B27, 10 μM DAPT (TargetMol), 30 μM Dexamethasone (Sigma-Aldrich), 10 μM SB431542 (TargetMol), and 10 μm Forskolin (TargetMol). Light microscopic images were captured with Nikon Ta2-FL.
3
Adipose-Derived Mesenchymal Stem Cell Senescence
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MSCs were isolated from discarded adipose tissue removed from healthy patients undergoing liposuction surgery, as described [22] . Informed consent was obtained from all patients. The procedure was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. The isolated cells were seeded in T75 asks containing 12 mL culture medium and incubated under humidi ed conditions at 37°C and 5% CO 2 . MSCs were continuously cultured to the third passage and then used for experiments. Cells were either left untreated or treated for 24 h with forskolin (60 µM, Target Mol, Boston, MA, USA). Pyrrolidine dithiocarbamic acid (PDTC, Beyotime, Shanghai, China) 100µM or adenylate cyclase inhibitor SQ22536 (Abcam, Cambridge, UK) 30µM were added to study their effects on MSCs senescence.
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