Rat pheochromocytoma (PC12) and SH-SY5Y cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA). PC12 cells were used for no more than 10 passages, and were in antibiotic-free DMEM supplemented with 10% horse serum and 5% FBS, whereas SH-SY5Y cells were grown in antibiotic-free DMEM supplemented with 10% FBS. Cells were maintained in a humid incubator (37°C, 5% CO2).
To isolate primary neurons, CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Animal Resource Center at Louisiana State University Health Sciences Center-Shreveport under specific pathogen-free conditions. All procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals. Fetal mouse cerebral cortexes of 14-18 days of gestation were used. Briefly, the pregnant mice were anaesthetized with pentobarbital and embryos were removed by a cesarean section under sterile conditions. Cerebral cortexes were isolated under dissection microscope and washed with ice-cold Ca2+/Mg2+-free Hank's buffered salt solution (HBSS) supplemented with 1% glucose and 50 μg/ml gentamicin. The isolated embryo cortexes were digested with papain solution [HBSS containing 500 μg/ml papain, 50 μg/ml DNase1, 1% glucose, 10 μM CaCl2, 5 mM EDTA] for 15 min at 37°C. Digested tissues were triturated into single cells using 1 ml pipette in NEUROBASAL™ Media supplemented with 2% B27 Supplement, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg/ml insulin, and 40 μg/ml of gentamicin. Isolated cells were seeded at a density of 2 × 106 cells/well in a 6-well plate coated with 10 μg/ml PDL in the above culture medium, and grown in a humid incubator (37°C, 5% CO2). Fresh medium was replaced every 3 days. The cells were used for experiments after 6 days of culture.
To isolate primary neurons, CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Animal Resource Center at Louisiana State University Health Sciences Center-Shreveport under specific pathogen-free conditions. All procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee and were in compliance with the guidelines set forth by the Guide for the Care and Use of Laboratory Animals. Fetal mouse cerebral cortexes of 14-18 days of gestation were used. Briefly, the pregnant mice were anaesthetized with pentobarbital and embryos were removed by a cesarean section under sterile conditions. Cerebral cortexes were isolated under dissection microscope and washed with ice-cold Ca2+/Mg2+-free Hank's buffered salt solution (HBSS) supplemented with 1% glucose and 50 μg/ml gentamicin. The isolated embryo cortexes were digested with papain solution [HBSS containing 500 μg/ml papain, 50 μg/ml DNase1, 1% glucose, 10 μM CaCl2, 5 mM EDTA] for 15 min at 37°C. Digested tissues were triturated into single cells using 1 ml pipette in NEUROBASAL™ Media supplemented with 2% B27 Supplement, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg/ml insulin, and 40 μg/ml of gentamicin. Isolated cells were seeded at a density of 2 × 106 cells/well in a 6-well plate coated with 10 μg/ml PDL in the above culture medium, and grown in a humid incubator (37°C, 5% CO2). Fresh medium was replaced every 3 days. The cells were used for experiments after 6 days of culture.
