Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons
Transcripción
To set up primary embryonic midbrain cultures from mouse embryonic day 13.5 mouse embryos, pool all of the harvested mid-brain floors into the same 1.5-milliliter tube, and wash the samples three times, with 500 microliters of calcium and magnesium-free HBSS per wash. After the last wash, replace the HBSS with 0.5% trypsin for a 30-minute incubation at 37 degrees Celsius.
At the end of the incubation, add 500 microliters of a freshly prepared DNase I in FBS solution to the partially digested tissue and use a siliconized glass pipette with a fire-polished tip to triturate the tissue. When only tiny, barely visible particles can be observed, allow these tissue fragments to settle to the bottom of the microcentrifuge tube, and transfer the supernatant into an empty 15-milliliter conical polypropylene tube.
Add one milliliter of HBSS to the tube containing DNase I in FBS, and mix several times by pipetting. Transfer one milliliter of this solution to the remaining tissue particles, and triturate the samples again. Then, pool the digested cell suspension with the tube of supernatant without transferring any remaining tissue fragments. After triturating the tissue sample one more time with the remaining DNase I in FBS in HBS, sediment the collected cells by centrifugation, and aspirate the supernatant without disturbing the pellet.
Wash the cells two times in two milliliters of warmed dopamine neuron medium per wash, and resuspend the cells at 3 x 104 cells per six microliters of fresh, warm medium concentration in a microcentrifuge tube. Next, remove the medium from each previously prepared micro-island and mix the cells with gentle pipetting before using a 10-microliter pipette to add six microliters of cells to each micro-island.
When all of the cells have been added, fill the empty wells at the edges of the plate with 150 microliters of water or PBS, and place the plate in the cell culture incubator for one hour. At the end of the incubation, add 100 microliters of fresh dopamine neuron medium to each well, and return the plate to the incubator.
