Primary cerebellar granule cells (CGCs) were treated with DMSO, Glutamate, Candesartan or Glutamate +candesartan
Growth protocol
Brains were dissected immediately and the cerebella were collected and placed in ice-cold Hank’s balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA). After removal of the meninges, the cerebella were dispersed into the same buffer containing 0.025% trypsin (Invitrogen) and digested for 15 min at 37ºC. Trypsin digestion was stopped by adding a same volume of Dulbecco’s Modified Eagle medium (DMEM) (Invitrogen), supplemented with 10% FBS (Invitrogen) and 0.1 mg/ml DNase I (Sigma-Aldrich, St. Louis, MO). After gentle trituration, digested tissues were centrifuged at 1000 X rpm for 5 min. The cell pellets were resuspended in the complete Neurobasal culture medium supplemented with 2% B27 (Invitrogen) and 0.5 mM GlutaMax (Invitrogen). After filtration through a 70 mm cell restrainer (BD Falcon, Vernon Hills, IL), cells were plated at a density of 1 X 106 cells/ml onto poly-L-lysine coated plates (Becton Dickinson and Company, Franklin Lakes, NJ) or chamber glass slides (Nalge Nunc International, Naperville, IL). Cultures were incubated in a humidified atmosphere of 5% CO2-95% air at 37ºC. Cytosine arabinofuranoside (Invitrogen) (10 mm) was added to the cultures 24 h after plating to arrest the growth of non-neuronal cells. Cultures 6 to7 days in vitro were used in this study.
Extracted molecule
total RNA
Extraction protocol
Trizol (Invitrogen) and RNaseazy (Qiagen) kits were used to extract total RNA according to the manufacturer's instructions.
Label
Biotin
Label protocol
Labeling was performed on a sample-by-sample basis according to manufacturer’s guidelines for use with the Affymetrix Rat HuGene-1_0-st-v1 GeneChips (Affymetrix, Inc). DNase treatment was included as part of isolation to remove possible contaminating DNA. Bioanalyzer nanochip (Agilent, Inc) and NanoDrop (ThermoFisher, Inc) were used to validate and quantitate the RNA prior to cRNA synthesis and labeling. 300 ng of total RNA was used for labeling with theAmbion WT Expression Kit (cat#4411974) and Affymetrix kit (Cat # 900652).After hybridization, the probe arrays were stained with streptavidin-phycoerythrin solution (Molecular Probes) and enhanced using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories).
Hybridization protocol
The hybridization cocktail containing the fragmented and labeled cDNAs were hybridized to Affymetrix Rat GeneChip 1.0 ST microarrays. The microarrays were washed and stained in an Affymetrix Fluidics Station using standard Affymetrix protocols.
Scan protocol
Genechips were scanned using an Affymetrix Gene Chip Scanner 3000.
Data processing
Gene expression intensities were calculated using GeneChip Command Console Software (AGCC). .cel files generated by the Affymetrix AGCC program were imported in the Affymetrix Expression Console software and RMA (Robust Multichip Analysis) normalization was performed to generate the .chp files.
