Dibutyryl cyclic amp dbcamp

Human bone marrow derived mesenchymal stem cell (hMSC) line (UE7T-13 cells, no. RBRC-RCB2161; RIKEN, Japan) [28 (link)–32 (link)], Growing and expansion media of hMSCs: Dulbecco’s Medified Eagle’s Medium (DMEM; Gibco) with 2mM _L-Glutamine, Fetal bovine serum (FBS; Gibco), Penicillin/Streptoycin (Gibco). Neuronal induction: dibutyryl cyclic AMP (dbcAMP; Sigma), 3-isobutyl-1-methylxanthine (IBMX; Sigma), human epidermal growth factor (hEGF; Sigma), recombinant human basic fibroblast growth factor (bFGF; R&D systems), fibroblast growth factor-8 (FGF-8; Pepro Tech), recombinant human brain-derived neurotrophic factor (BDNF; R&D systems), and nerve growth factor (NGF), Neurobasal medium (Gibco) supplemented with 2% B27 supplement (Gibco), 2 mM l-glutamine (Gibco).

SN56 neuroblastoma cells were grown in DMEM, and Neuro-2a neuroblastoma cells were grown in MEM. Media was supplemented with 10 % (v/v) heat-inactivated FBS (Gibco), and 100 μg/mL penicillin/streptomycin (Gibco). Cells were kept in 25 cm² or 75 cm² flasks (Falcon) in an incubator at 37 °C in a humidified atmosphere containing 5 % CO₂ and were split every 3–4 days. Cells were seeded at a density of 4 × 10⁵ cells/35-mm dish (MatTek, MA, USA) one day prior to transfection date. Cells were then transiently transfected with Lipofectamine 2000 or TurboFect according to manufacturer's instructions (Invitrogen, MA, USA). Following an incubation period of 24 h, N2a cells were differentiated by serum withdrawal; SN56 cells were also treated with 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma, MO, USA) [20 (link)].

Fully grown oocytes containing a visible intact germinal vesicle (GV) and a diameter greater than 70 μm were collected from post-natal day (PD) 19 females at the GV stage. Ovaries were dissected into several fragments and incubated at 37 °C in air in minimal essential medium (MEM, Life Technologies) buffered at pH 7.2 using HEPES (MEM-H), containing sodium pyruvate (0.25 mM, Sigma), penicillin G (63 mg/L, Sigma), streptomycin (50 mg/L, Sigma), and bovine serum albumin (BSA, 1 mg/mL, Sigma). Large follicles were punctured using fine needles. The collection media was supplemented with dibutyryl cyclic AMP (dbcAMP, 0.1 mg/ml, Sigma) to prevent resumption of meiosis. Oocytes were collected using a mouth-controlled micropipette, transferred to fresh medium, and the diameter was measured using an ocular micrometer. To obtain oocytes that had undergone germinal vesicle breakdown (GVBD) and completed maturation to metaphase II, respectively, GV-stage oocytes were transferred to MEM-NAHCO₃ containing pyruvate, antibiotics and BSA, and incubated at 37 °C in 5% CO₂ in air for 5 hr or overnight, respectively, to allow meiotic maturation. GVBD oocytes were identified by the absence of the GV and mature oocytes were by the presence of a polar body.

Horseradish peroxidase-conjugated secondary anti-IgG antibodies were purchased from Millipore (Billerica, MA, USA). Alexa Fluor 647-conjugated secondary antibody was obtained from Molecular Probes (Eugene, OR, USA). Polyclonal rabbit anti-HSV-1 antibodies were from Dako and Antibodies Online GmbH. Mouse anti-PLP MAB388 and anti-integrin β-1 MAB5199 antibodies were from Millipore. Anti-CNPase C5922, low-glucose DMEM, fetal bovine serum (FBS), human insulin, triiodothyronine (T3), apo-transferrin, sodium selenite, putrescine, dibutyryl cyclic AMP (dbcAMP), and protease inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-CD63 antibody 1B5 (87 (link)) was a kind gift from A. Fraile-Ramos (Universidad Complutense, Madrid, Spain). Anti-CD81 antibody 5A6 (SC-23962) was a kind gift from F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain). The rabbit LC3-II antibody was from Novus Biologicals. Anti-flotillin-1 antibody 610821 was from BD Biosciences. Mowiol was from Calbiochem (Merck Chemicals, Germany).

NCSCs were cultured on polyornithine‐laminin‐fibronectin‐coated culture dishes in DMEM/F12 medium supplemented with 1× N2, 10 ng/mL brain‐derived neurotrophic factor (BDNF) (PeproTech), 10 ng/mL glial cell‐derived neurotrophic factor (GDNF) (PeproTech), 10 ng/mL nerve growth factor (NGF) (PeproTech), 200 μmol/L ascorbic acid and 0.1 mmol/L dibutyryl cyclic AMP (dbcAMP) (all from Sigma‐Aldrich) for 10‐14 days. The medium was changed every other day and assayed by immunocytochemistry.

The VM was dissected from female SD rat embryos at E14. Single cells were isolated from the tissue and plated on PLO/FN-coated glass slides in 24-well plates. Cells were cultured in N-2 growth medium containing 20 ng/mL bFGF. The differentiation medium consisted of N-2 supplemented with 0.2 mM ascorbic acid and 250 μg/mL dibutyryl cyclic AMP (db-cAMP; Sigma-Aldrich).

All experimental procedures applied to animals were approved by the Home Office (UK) or by the Local Ethical Committe no. 1 (Warsaw, Poland), and were performed in compliance with the national regulations. Mouse F1 (C57Bl6 x CBA) females were superovulated by an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG, Intervet) followed 48 hrs later by 10 IU of human chorionic gonadotrophin (hCG, Intervet) and mating with F1 males. Zygotes and 2-cell embryos were recovered from oviducts into M2 medium 16–20 hrs and 44 hrs after hCG injection, respectively. MII oocytes were recovered 15 hrs post hCG injection from oviducts of unmated females into M2 medium. GV oocytes were recovered from ovaries 48 hrs after the PMSG injection into M2 medium supplemented with 150 μg/ml dibutyryl cyclic AMP (dbcAMP, Sigma-Aldrich).