HT-22 hippocampal neuronal-like cells (provided by Prof. Culmsee) were used in this study. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Cramlington, UK, #42340-025) containing 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Logan, UT, USA, #SV30160.03), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco; Life Technologies, Carlsbad, CA, USA, #15070-063) and 1% sodium pyruvate (Gibco, Life Technologies Corporation, Carlsbad, CA, USA, #11360-070). HT-22 cells were incubated at 37 °C and 5% CO2 and treated, where indicated, with erastin (Tocris), CE3F4 (IC50 = 23 ± 3 µM) [24 (link)], ESI-05 (IC50 = 0.4 µM) [25 (link)] (BioLoG, Bremen, Germany, Cat.No. M 092), ESI-09 (IC50 = 3.2 µM for Epac1 and 1.4 µM for Epac2) [26 (link),27 (link)] (BioLog, Bremen, Germany, Cat.No. B 133), 8-PCPT-2-O-Me-cAMP (AC50 = 1.8 µM) (BioLog, Bremen, Germany, Cat.No. C 041). Only mycoplasma free cells and passage numbers between 300–400 were used for the experiments.
8 pcpt 2 o me camp
Manufactured by Biolog
Sourced in Germany
8-pCPT-2'-O-Me-cAMP is a chemical compound used in laboratory research. It functions as a cAMP analog that selectively activates the cAMP-dependent protein kinase Epac.
Automatically generated - may contain errors
Lab products found in correlation
6 bnz camp, by Biolog (4 mentions)
Forskolin, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Ce3f4, by Bio-Techne (1 mentions)
Erastin, by Bio-Techne (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Isoprenaline, by Merck Group (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Omniscript reverse transcriptase, by Qiagen (1 mentions)
8 protocols using 8 pcpt 2 o me camp
1
Neuronal-like Cell Culture and Treatment
2
Pharmacological Regulation of Cell Signaling
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Formoterol was a gift from AstraZeneca (Lund, Sweden) and olodaterol from Boehringer Ingelheim (Biberach, Germany). All other drugs were purchased: actinomycin D, cholera toxin, cycloheximide, forskolin, isoprenaline, IBMX (2-isobutyl-1-methylxanthine), orciprenaline, penicillin-streptomycin solution, and trypsin from Sigma (Deisenhofen, Germany); ICI 118,551 ((±)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol hydrochloride) from Biozol (Eching, Germany); 6-Bnz-cAMP (N6-benzyladenosine-3′,5′-phosphate) and 8-pCPT-2′–O-Me-cAMP (8-(4-chlorophenylthio)-2′–O-methyladenosine-cAMP) from Biolog Life Science Institute (Bremen, Germany); desoxynucleotide mixture from Fermentas (St. Leon-Rot, Germany); Eagle’s minimal essential medium (MEM) with Earl’s salts and L-glutamine, non-essential amino acids from PAA (Cölbe, Germany); fetal calf serum (FCS) from Biochrom (Berlin, Germany); Taq DNA-polymerase from Invitrogen (Karlsruhe, Germany); and Omniscript reverse transcriptase, RNeasy Mini kit, QuantiTectTM SYBR Green PCR kit, and RNase-free DNase set from Qiagen (Hilden, Germany). Oligodesoxynucleotides for qPCR were obtained from Eurofins MWG Operon (Ebersberg, Germany).
3
Inducing Stumpy Trypanosome Forms for Mosquito Feeding
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The pleomorphic T.b. brucei AnTat1.1E bloodstream forms were cultured in HMI9 medium as described above. In order to obtain stumpy forms, the parasite stage infective to the insect host, an in vitro induction was performed in slender forms cultured at 1.105 parasites/mL with the cAMP analogue 8-pCPT-2′-O-Me-cAMP (Biolog, Germany) at 5μM for 48h [26 (link)]. The presence of stumpy forms was verified by using the anti-PAD1 rabbit polyclonal antibody (1:250, Keith Matthews, Edinburgh, UK) targeting the carboxylate-transporter Proteins Associated with Differentiation 1 (PAD1) [27 (link)]. Differentiated parasites were centrifuged and resuspended in commercial mechanically defibrinated sheep blood (BCL, France) at 108 parasites/mL in order to feed Anopheles females through a membrane feeding system in which the blood mixture is maintained at 37°C. Mosquito guts were collected 48h and 5 days post-trypanosome ingestion. These experiments were performed to remove any potential confounding factors from the blood of infected mice and be able to attribute results to the direct effect of trypanosomes on the mosquitoes.
4
Regulations of Inflammatory Cytokine Secretion
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RAW264.7 cells and HEK293 cells are obtained from ATCC, and maintained in DMEM (Dulbecco′s modified Eagle′s medium) supplemented with 10% fetal bovine serum (FBS) from Gibico. BSA (bovine serum albumin) and anti-BSA antibody (α-BSA) are obtained from Invitrogen and MP Biomedicals, respectively. Dimethyl sulfoxide (DMSO) is obtained from Sigma-Aldrich. Rolipram and Roflumilast are obtained from Cayman Chemical and APExBIO, respectively. 6-Bnz-cAMP (PKA agonist) and 8-pCPT-2′-O-Me-cAMP (Epac agonist) are obtained from Biolog Life Science Institute. PKA inhibitor H-89 is obtained from Beyotime Biotechnology. ELISA kits for measuring cAMP, TNF-α, MIP-1α, MIP-1β, MIP-2 and KC are all obtained from R&D Systems. ELISA kit for measuring mouse albumin is obtained from Bethyl Laboratories.
5
Endothelial cell permeability assay
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Endothelial cells (1.0 × 105/cm2) were cultured on polycarbonate cell culture inserts (pore size 0.4 µm, porosity 0.9·108/cm2; Nunc, ThermoFisher, Waltham, CA) coated with 0.1% gelatin for 48 h. When appropriate, cultures were serum starved for 24 h and cells were treated with fenoterol (1 µM; Boehringer Ingelheim, Germany), forskolin (10 µM; Tocris, UK), 6-Bnz-cAMP (300 µM) or 8-pCPT-2′-O-Me-cAMP (100 µM) (Biolog Life Science Institute, Germany). Parallel cultures were maintained under normoxic (21% oxygen tension) and hypoxic conditions (2% oxygen tension) for 48 h prior to experiments. Permeability was assessed by the addition of 10 µg/ml FITC-dextran in the upper compartments, and fluorescence in the lower compartments was assessed on a spectrofluorescence reader at Ex485/Em519 after 30 min.
6
Reagents Used in Calcium Signaling Experiments
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H89, NKH 477, 8-Br-cAMP, and 8-Br-cGMP were from R&D Systems (Abingdon, Oxford, UK). Sp-cAMPS, 6-Bnz-cAMP, 8-pCPT-2′-O-Me-cAMP, Rp-cAMPS, Rp-8-CPT-cAMPS, ESI-09, and HJC0197 were from Biolog (Bremen, Germany). Ionomycin, SQ 22536, DDA and myristoylated-PKA inhibitor peptide (PKI) were from Merck-Millipore (Watford, UK). A membrane-permeant peptide inhibitor of A kinase-anchoring proteins (AKAPs) [stearated Ht31 AKAP inhibitor peptide (st-Ht31)] and its proline-modified inactive form (st-Ht31P) were from Promega (Southampton, UK). Thapsigargin was from Alomone Laboratories (Jerusalem, Israel). PAR1 peptide, histamine dihydrochloride, forskolin, IBMX, and PGE2 were from Sigma-Aldrich (Welwyn Garden City, UK). Butaprost (free acid) and L902,688 were from Cayman Chemicals (Ann Arbor, MI). Membrane-permeant caged IP3 (ci-IP3PM) was from SiChem (Bremen, Germany). [2,8-3H] adenine ci-IP3PM, d -2,3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl) ester was from Perkin Elmer (Seer Green, Bucks, UK). Fluo-8 was from Stratech Scientific Ltd (Newmarket, Suffolk, UK). Other reagents were from Sigma-Aldrich, sources specified previously (Pantazaka et al., 2013 (link)) or identified in this section.
7
Autophagy and Oxidative Stress Assays
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Forskolin (#F6886), doxorubicin (#D1515), propidium iodide (PI) (#P4170), Ruthenium Red (RR) (#557450) and EDTA (#03690) were purchased from Sigma-Aldrich. CYTO-ID Autophagy detection kit ver. 2.0 (ENZ-KIT175) was purchased from Enzo Life Sciences. 8-CPT-cAMP (#C010-500) and 8-pCPT-2′-O-Me-cAMP (#C041-25) were obtained from BioLog, whereas N-acetyl-L-cysteine (#A9165), Fluo-3 (F1242), Indo-1 (I1223) and CellROX Green Oxidative Stress Reagent (C10444) were purchased from ThermoFisher. JC-1 (#420200) stain was purchased from Calbiochem. BAPTA-AM (#ab120503) and the EPAC1 rabbit monoclonal antibody (#ab109415) were obtained from Abcam, and the antibody was used at a final dilution of 1:1000. The GAPDH rabbit monoclonal antibody (#5174) and the rabbit polyclonal antibody recognizing cleaved-PARP (#9541S) were both purchased from Cell signaling technology, and they were used at a final dilution of 1:5000 and 1:1000, respectively.
8
Modulation of Trypanosoma brucei Differentiation
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