age: Gestation day 15 strain: Swiss Albino Mouse cell type: Murine primary cortical neurons age of culture: Day 6 ogd treatment: 0hr 24hr reperfusion: +
Treatment protocol
Primary neuronal cultures from day 6 were subjected to oxygen-glucose deprivation (OGD) conditions as previously described (Ziu et al. 2011). Glucose-free Earle’s balanced salt solution (EBSS) was saturated with a mixture gas of 5% CO2, 95% N2, in a ProOx in vitro chamber (BioSpherix, USA) at 37ºC overnight, with O2 maintained at 0.1%. Day 6 neuronal cultures were washed twice with this medium and incubated for 2, 4, 6, 8hr in the chamber. OGD was terminated by replacing the glucose-free EBSS with reperfusion medium (Neurobasal medium with L-glutamine and Penicillin-streptomycin, without B27 supplement). Control cultures were treated identically, but without exposure to OGD conditions. During reperfusion, the cells were maintained in a regular 5% CO2 incubator for 24hrs.
Growth protocol
Primary cultures of cortical neurons were established from E15 Swiss albino mouse brains. The cortices were dissected from E15 mouse embryos and washed with Hanks’ balanced salt solution (HBSS, 14025-092, Gibco, Invitrogen, USA). The cortical slices was dissociated with 0.05% (w/v) trypsin in HBSS without Ca2+/Mg2+ (14175-095, Gibco, Invitrogen, USA) for 30 min at 37 °C and neutralized with 1mg/ml trypsin inhibitor (T6522, Sigma, USA). Single cells were obtained by gentle trituration in Neurobasal medium (21103-049, Gibco, Invitrogen, USA) supplemented with B27 (17504-044, Invitrogen, USA), L-glutamine and Penicillin-streptomycin (Gibco, Invitrogen, USA). The cells were counted by trypan blue exclusion and seeded on to poly-d-lysine coated 24 well plates at a density of 120, 000 cells/cm2. Cultures were maintained at 37 °C with 5% CO2 in a tissue culture incubator. Cells were harvested on Days 2, 4, 6, 8.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cells by a single-step method using Trizol (Invitrogen, Life Technologies, USA) according to the manufacturers’ protocol. The concentration and integrity of the RNA were determined using Nanodrop ND-2000c spectrophotometry (Nanodrop Tech, Rockland, Del) and denaturing polyacrylamide gel electrophoresis, respectively. Thereafter, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre, USA).
Label
Cy3
Label protocol
Agilent Quick Amp Labeling Kit was used for sample labeling.
Hybridization protocol
Hybridization was performed in Agilent’s SureHyb Hybridization Chambers.
Scan protocol
Slides were scanned with the Agilent DNA Microarray Scanner. Data was extracted using Agilent Feature Extraction software.
Data processing
Raw signal intensities were normalized in quantile method by GeneSpring GX v11.5.1, and low intensity mRNAs and lncRNAs were filtered.
