Bitan:Isolation of primary cells

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Dissection of the hippocampus from E18 embryo Check all materials are ready? Compete all procedures within 2 hours! Before hippocampal dissection 1. Sterilize all the equipments by autoclave 2. Coat the plate or cover slip with poly D-lysine solution (0.1%) and allow at least 2 h in culture incubator. After coating wash the plate or cover slips with sterile water at least 3 times. Hippocampal dissection 1. Euthanized pregnant rat or mouse with CO2 in desiccators (for 2 min) 2. Cut skin and remove uterus 3. Place in an ethanol-wiped dish and take fetuses out 4. Decapitate fetuses, dissect out the brains cutting along the sagital, frontal and place them in ice-cold Lebovitz’s L15 media and immediately take those to under laminar hood. 5. Using small forcep, cissor remove meanings and dissect out hippocampi under dissecting microscope. 6. Place hemisphere of brain and hippocampi in ice-cold Lebovitz’s L15 media 7. Place them in a 15-ml centrifuge tube, and bring the total volume to 4.5ml with Lebovitz’s L15 media 8. Add 0.5 ml of trypsin (0.25%) (10 fold) and incubate at 37oC for 30 min to 1h 9. Dissociate the tissue with fire polished Pasteur pipette (30-40 times up and down) or continue pipetting until no lumps of tissue remain 10. Add culture medium and count cell density 11. Place in coated dish with your favorable density 12. Replace culture medium with fresh one, once or twice a week Culture of cells Note: do not dry the cell surface in medium change! 1. Replace culture medium with fresh DMEM containing 10% heat inactivated horse serum after 2 or 3 days 2. Replace culture medium with fresh DMEM containing 10% heat inactivated horse after 1 week 3. To reduce glial proliferation, add cytosine arabinoside 5μM after 2 or 3 days of culture 4. After 6 days of culture it is ready for toxicity study

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