A Technique to Culture Mouse Hippocampal Neurons
Transcrição
To begin, dissect six to eight hippocampi from postnatal, one-day-old pups with HBSS medium in a Petri dish. Chop the hippocampi with dissection scissors into smaller pieces, and transfer them from the dish to a 15-milliliter tube. Wash the hippocampi twice with 5 milliliters of HBSS, and leave them in 4.5 milliliters of HBSS after the wash.
Add 0.5 milliliters of 2.5% trypsin into 4.5 milliliters of HBSS, and incubate in a 37-degree Celsius water bath for 15 minutes, inverting the tube every 5 minutes. Well-digested hippocampi should become sticky and form a cluster. If needed, extend the digestion for 5 more minutes.
Wash hippocampi with HBSS three times for 5 minutes per wash. After the wash, add 2 milliliters of HBSS, and pipette the hippocampi up and down with a Pasteur pipette 15 times. Triturate the tissue with a fire-polished Pasteur pipette 10 times, and rest the tube for five minutes until all chunks set to the bottom. Repeat the trituration with the remaining chunks until most of them have disappeared.
Then, use a 1-milliliter pipette tip to gently transfer the supernatant containing the dissociated neurons to plating dishes. Add it directly to the pre-incubated plating medium and shake the plate gently. 2 to 4 hours after seeding, check the plating dishes with a light microscope. The majority of the neurons should have attached to the coverslip.
Using a fine-tipped forceps, flip the coverslips to the glial cell feeder dishes containing pre-conditioned neuronal culture medium with the wax dot side facing downwards. Neurons can grow in the glia cell feeder dishes for up to one month. Feed neurons every seven days with 1 milliliter of fresh neuronal culture medium.
