Erythropoietin is a glycoproteic hormone that regulates hematopoiesis by acting on its specific receptor (EpoR). oxidative stress in the CNS such as Alzheimer disease. (DIV) were treated with Epo or EpoL at neuroprotective concentrations in co-incubation with A oligomers at 37?C and 5% CO2. 2.4.3. Epo pre-treatment assay PC-12?cells at 85% confluence were pretreated with Epo or EpoL for 1?h and then stressed with A40 peptide oligomers during 24? h using the same medium with Epo or EpoL. Subsequently, the percentage of live cells was quantified. 2.5. Soluble oligomers of A The human A1C40 was dissolved in Dimethyl sulfoxide (DMSO, Sigma- Aldrich) at a concentration of 80?M and stored at ?20?C. The soluble oligomer solution was freshly prepared from a stock external solution and aggregated under standard conditions of 200?rpm?at 37?C for 2?h [28]. The final concentrations obtained for oligomers were 0.5?M, 1?M, and 5?M. The oligomer species were previously described and confirmed [29,30]. Cells were treated for 24?h alone or co-incubated with Epo or EpoL extracts at different concentrations. The A25-35 peptide was obtained from Sigma (Madrid, Spain), dissolved in phosphate buffer solution (PBS) and incubated with organotypic hippocampus cultures at different concentrations, according to the protocol previously described [31]. 2.5.1. Cell viability assays PC-12?cell cultures were seeded at a density of 90,000?cells/well and used 24?h after plating. After exposing the cells to each experimental condition, they were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1?mg/ml) for 30?min, and, thereafter, precipitated MTT was dissolved using isopropanol cooled for 15?min. The tetrazolium ring of MTT can be cleaved by active dehydrogenases in order 868049-49-4 to produce a precipitated formazan compound. Absorbance was measured in a multiplate reader (NovoStar, LabTech BMG, Germany) at two wavelengths: 560?nm and 620?nm, and the difference was quantified using NovoStar Software for the different experimental conditions. 2.5.2. Quantitative real time PCR Total RNA of PC-12?cells treated was purified using TRIZOL (Sigma, USA), and the reaction was performed with the commercial kit KAPA SYBR 868049-49-4 FAST qPCR (KapaBiosystems, USA) and the equipment for HDAC2 Stratagene MX3000P (ThermoFisher, USA) real-time PCR. The qPCR was performed using RNA as 868049-49-4 a template, and the primers were ordered from Integrated DNA Technologies (Coralville, USA): BcL-2 (Forward: GATGACTGAGTA CCTGAACCG, Reverse: CAGAGACAGCCAGGAGAAATC) and -actin (Forward: CACTTTCTACAATGAGCTGCG, Reverse: CTGGATGGCTACGTACATGG). The comparative threshold cycle values were normalized for the -actin reference gene and the results were expressed as CT relative quantification by the 2-CT method. 2.6. Organotypic hippocampal cultures Organotypic hippocampal cultures were obtained from brains of 8C10 day old Sprague Dawley rats. Hippocampal slices (300?m thick) were prepared and separated in ice-cold Hank’s balanced salt solution (HBSS) composed of: glucose 15?mM, CaCl2 1.3?mM, KCl 5.36?mM, NaCl 137.93?mM, KH2PO4 0.44?mM, Na2HPO4 0.34?mM, MgCl2 0.49?mM, MgSO4 0.44?mM, NaHCO3 4.1?mM, HEPES 25?mM, 100 U/ml of penicillin, and 0.100?mg/ml of gentamicin. Four slices were placed on Millicell 0.4?m tradition inserts (Millipore, Spain) within every well of the six well tradition plate with tradition media. The tradition media was made up of 50% minimal important moderate (MEM), 25% Hank’s well balanced salt remedy, and 25% heat-inactivated equine serum (Existence Systems, Spain). After 4 times in tradition, the slices had been treated with -amiloyd25-35 (Sigma-Aldrich, Spain) for 4 times, with or without Epo or EpoL at previously established neuroprotective concentrations (Castillo et al., 2018). Ethnicities had been taken care of at 37?C and 5% CO2, as well as the moderate was changed weekly twice. 2.6.1. Cell loss of life dimension of organotypic ethnicities At the ultimate end of every test, organotypic cultures had been packed with 1?g/ml propidium iodide (PI) and Hoechst 33342 (Hoechst) for 30?min?at 868049-49-4 37?C and 5% CO2. PI and Hoechst fluorescence through the cornu Ammonis 1 (CA1) area was measured utilizing a Fluorescence inverted NIKON eclipse T2000-U microscope. Wavelengths of emission and excitation for PI and Hoechst had been 530 or 350, and 580.