Cyclic AMP, cyclic GMP, the other nucleotides, (3-mercaptopropyl) trimethoxysilane and Escin were purchased from Sigma. Nickel (II) sulfate hexahydrate IBMX, ibudilast, and forskolin were purchased from Fujifilm. Maleimido-C3-NTA was purchased from Dojindo. Potassium methanesulfonate was purchased from Tokyo Chemical Industry. Fluorescein reference standard was purchased from Invitrogen.
Cyclic amp
Manufactured by Merck Group
Sourced in United States
Cyclic AMP is a laboratory product that functions as a crucial intracellular secondary messenger. It plays a central role in various cellular signaling pathways, mediating the effects of numerous hormones and neurotransmitters.
Automatically generated - may contain errors
Lab products found in correlation
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (5 mentions)
Ascorbic acid, by Merck Group (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
Hydrocortisone acetate, by Merck Group (3 mentions)
Cyclic gmp, by Merck Group (2 mentions)
Rat tail collagen type 1, by BD (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Retinoic acid, by Merck Group (2 mentions)
Penicillin streptomycin amphotericin, by Thermo Fisher Scientific (2 mentions)
Fluorescein reference standard, by Thermo Fisher Scientific (1 mentions)
Escin, by Merck Group (1 mentions)
Maleimido c3 nta, by Dojindo Laboratories (1 mentions)
Dulbecco s modified eagle medium dmem, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Egm 2 mv medium, by Cambrex (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Cambrex (1 mentions)
16 protocols using cyclic amp
1
Synthesis and Characterization of Biomolecular Probes
2
Isolation and Culture of Human LECs
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Lymphatic endothelial cells (LECs) were obtained through isolation from human neonatal foreskins via immunomagnetic separation using the LEC marker podoplanin as described previously [48] (link). The LECs were expanded in T75 flasks that had been previously coated for 1 h with a collagen solution containing type I rat tail collagen (BD Biosciences, San Jose, CA) at a concentration of 50 µg/mL in 0.1% acetic acid (Sigma). The cells were grown in EBM (Lonza, New York) supplemented with 20% FBS (Atlanta Biologicals), 1% Glutamax, 1% Pencillin-Streptomycin-Amphotericin (Gibco), 25 mg/mL cyclic-AMP, and 1 mg/mL hydrocortisone acetate (both from Sigma). LECs were split at 80–90% confluence and were used in experiments either at passage 8 or 9. Human dermal fibroblasts (HDFs) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% FBS and 1% Pencillin-Streptomyacin-Amphotericin. HDFs were split at 80–90% confluence and were used in experiments at passage 14.
3
Decidualization and Hormonal Regulation of HESCs
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Aliquots of frozen primary HESCs previously isolated and characterized, as described [40 (link)], were thawed and cultured in basal media containing Dulbecco’s MEM/F12 with 10% stripped calf serum and 1% antibiotic (Life Technologies, Carlsbad CA, USA). To induce decidualization, confluent cultures were incubated with 10−8 M E2 (Sigma-Aldrich) + 10−7 M MPA (Sigma-Aldrich) and 5 × 10−5 M cyclic AMP (Sigma; named EMC media) for 0, 3, or 6 days. In parallel, cultures were incubated in basal media containing 10−8 M E2 alone, or in combination with 10−7 M ORG (Merck & Co, Whitehouse Station, NJ, USA), or 10−7 M ETO (Sigma-Aldrich), or 10−7 M LNG (Sigma-Aldrich), or 10−7 M MPA, or 10−7 M DEX (Sigma-Aldrich) for 7 days. Then, cultures were switched to defined medium as described previously [23 (link)] with corresponding steroids for 6 h or 24 h for RNA and protein analysis, respectively.
Frozen banked HEECs isolated and characterized, as described [13 (link),74 (link)], were thawed and cultured in EGM-2 MV Medium with 5% fetal calf serum (Cambrex Bio Science, Walkersville, MD, USA). Similar to the HESCs’ treatment, confluent HEECs seeded in 6-well plates were incubated with 10−8 M E2 (as a control) ± 10−7 M ORG, or 10−7 M ETO, or 10−7 M LNG, or 10−7 M MPA, or 10−7 M DEX for 7 days. After serum starvation, HEECs were treated with the corresponding steroids for 6 h for RNA extraction.
Frozen banked HEECs isolated and characterized, as described [13 (link),74 (link)], were thawed and cultured in EGM-2 MV Medium with 5% fetal calf serum (Cambrex Bio Science, Walkersville, MD, USA). Similar to the HESCs’ treatment, confluent HEECs seeded in 6-well plates were incubated with 10−8 M E2 (as a control) ± 10−7 M ORG, or 10−7 M ETO, or 10−7 M LNG, or 10−7 M MPA, or 10−7 M DEX for 7 days. After serum starvation, HEECs were treated with the corresponding steroids for 6 h for RNA extraction.
4
Neural Differentiation of NPCs
5
Differentiating Human ESCs into Mature Motor Neurons
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All human ESC experiments were conducted with prior approval from the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee. H9 human ESCs (Madison, Wisconsin) were maintained on irradiated MEFs in human ESC media (DMEM/F12, 20% knockout serum replacement, 1% nonessential amino acids, 1% glutamax, 0.1 mM 2-mercaptoethanol and 10 ng ml−1 of basic fibroblast growth factor). Human ESCs were differentiated to MNs as previously described14 (link). RA was added to the human ESC-derived neural progenitors after 10 days of differentiation. Neural rosettes were manually isolated and transferred into neural differentiation media with 0.1 μM RA and 1 μM SAG. After 28 days, human ESC-derived MN progenitors were cultured in human MN maturation media with 0.1 μM RA, 50 nM SAG, cyclic AMP (0.1 μM; Sigma), ascorbic acid (200 ng ml−1; Sigma), insulin growth factor 1 (10 ng ml−1; Peprotech) and NFs (GDNF, BDNF and CNTF; all 10 ng ml−1; Peprotech)). For immunostaining, hESC-derived MNs were lightly dissociated using 20% TrypLE (Invitrogen) and plated on matrigel-coated glass coverslips in MN maturation media.
6
Antibodies Production and Characterization
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Anti–coronin A antibodies were raised in rabbits (Eurogentec, SA, Seraing, Belgium) against a synthesized peptide corresponding to a unique C-terminal portion of the coronin A amino acid sequence (GGFVKKASAVEFKPV, residues 388–402) using a 28-d immunization protocol. The antibody was purified from the serum of immunized rabbits by affinity chromatography using the same peptide as affinity matrix. Anti-Gα2 was raised in rabbits (Thermo Scientific, Waltham, MA) against a synthesized peptide (CASSMEGEKTNTDINLSIEK, residues 4–24; Kumagai et al., 1989 (link)) using a 70-d immunization protocol. The mouse monoclonal antibody against CsA (123-353-1) and anti–discoidin 1 (80-52-13) developed by G. Gerisch (Max Planck Institute of Biochemistry, Martinsried, Germany) were obtained through the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA) and the National Institute of Child Health and Human Development. Mouse anti-actin clone c4 was purchased from Millipore (Temecula, CA). The cyclic AMP and the 8-bromo-cAMP analogue were obtained from Sigma-Aldrich (St. Louis, MO). Latrunculin A was from Calbiochem (Billerica, MA). Antibodies against CMFR1 were a kind gift from Richard Gomer, Texas A&M University (College Station, TX) and were purified using a protein A column (HiTrap protein A HP; GE Healthcare, Uppsala, Sweden).
7
Thermodynamic Binding of CxD7 Ligands
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Thermodynamic binding parameters of CxD7L1 and CxD7L2 to several pro-hemostatic ligands were tested using a Microcal VP-ITC microcalorimeter. The panel of substances tested included several nucleosides/nucleotides or derivates (ATP, ADP, 5′-AMP, 3′-AMP, cyclic AMP, adenosine, GTP, TTP, inosine, sodium polyphosphate, Sigma-Aldrich), biogenic amines (epinephrine, norepinephrine, histamine, serotonin, Sigma-Aldrich), and pro-inflammatory/pro-hemostatic lipid compounds (LTB4, LTC4, LTD4, LTE4, arachidonic acid, and the stable analog of TXA2: U-46619, Cayman Chemicals). Ligands and protein solutions were prepared in 20 mM Tris–HCl, pH 7.4, 150 mM NaCl (TBS) at 30 and 3 µM, respectively. Lipid ligands were prepared by evaporating the ethanol or chloroform solvent to dryness under a stream of nitrogen. Lipid ligands were further dissolved in TBS and sonicated for 10 min (Branson 1510) to ensure dissolution. Lipid ligands were used at 50 µM of ligand and 5 µM of protein. Injections of 10 µL of ligand were added to the protein samples contained in the calorimeter cell at 300 s intervals. Experiments were run at 30 °C. Thermodynamic parameters were obtained by fitting the data to a single-site binding model in the Microcal Origin software package version 7 (OriginLab). For saturation studies, CxD7L2 protein was pre-incubated with 50 µM serotonin for 30 min and titrated with LTD4.
8
Differentiation of iPSCs to Neurons
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Differentiation of iPSCs into neural progenitors was performed by treatment of iPSCs with 3μM glycogen synthase kinase 3 (GSK3) inhibitor (CHIR99021) and 2μM TGFβ inhibitor (SB431542) as previously described, with neural progenitors appearing in culture after seven days. For neuronal differentiation, neural progenitors were seeded at a density of 150 000 cells/well onto a Matrigel-coated six-well plate in neural induction medium [25 (link)]. After two days, medium was changed to neuronal differentiation medium (DMEM/FBS supplemented with N2, B27, 300ng/ml cyclic AMP (Sigma), 0.2mM ascorbic acid (Sigma), 10ng/ml BDNF (Peprotech) and 10ng/ml GDNF (Peprotech)) and the cells maintained in culture for 14 to 21 days, with neurite outgrowth typically observed after one week of culture. Medium was changed every 2–3 days. Characterisation was performed by immunocytochemistry and qRT-PCR (for antibodies and primers see S1 –S3 Tables).
9
E. coli Growth and Genetic Manipulation
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E. coli K-12 strains (SI Appendix, Table S1 ) were grown aerobically at 37 °C in LB medium (63 (link)) with shaking at 200 rpm. Colony formation was on LB agar at 37 °C. Bacteriophage P1-mediated transduction (64 ) or CRISPR-based allelic exchange (24 (link)) was used for strain construction. Flow cytometry reagents were purchased from Becton Dickinson. Tryptone, yeast extract, powder for LB broth and agar, and carboxy-H2DCFDA were obtained from Thermo Fisher Scientific. Other reagents, including antimicrobials (ciprofloxacin, oxolinic acid, kanamycin, ampicillin, tetracycline, moxifloxacin, and chloramphenicol), phenol, dimethyl 2-oxoglutarate, cyclic AMP, sodium pyruvate, and DMSO were purchased from Sigma-Aldrich. Gentamicin, amikacin, hydrogen peroxide, chlorhexidine, ethanol, isopropanol, 1-butanol, potassium dichromate, sodium hypochlorite solution (5.2%), hydrogen chloride, and sodium hydroxide were purchased from Sangon Biotech. Meropenem (Shenghuaxi Pharmaceutical) and ceftriaxone (Roche) were from Zhongshan Hospital (Xiamen, China) pharmacy.
10
Karyotyping and Differentiation of iPSC-NSCs
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G-banded karyotyping of iPSC-NSCs (p9) was performed by Cell Line Genetics. Terminal in vitro differentiation was carried out in Neurobasal:DMEM/F12 (GIBCO) containing N-2 (1:100), B-27 (1:100), and cyclic AMP (300 ng/ml; Sigma) for 1 month. Quantification of the percentage of undifferentiated and terminally differentiated iPSC-NSCs expressing cell-type specific markers was performed by manual counting of immunofluorescently labeled slides. Percentages are represented as means ± SEM (n = 4).
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