Hippocampal Neuron Cultures
实验概要
The protocol provides a method of hippocampal neuron cultures.
主要试剂
Begin by timing the pregnant mouse at E17-E19 days of gestation. Have ready the following:
1. Cold Hank’s balanced salt solution
2. 1x trypsin (0.25% in HBSS)
3. 1x soybean trypsin inhibitor (1 mg/ml)-SBTI
4. Neurobasal medium with:
1) B27 supplement (2 ml per 100 ml medium)
2) L-glutamine (0.5 mM)
3) L-glutamate (25 mM)
4) Gentamicin (10 g/L)
This is called “G3” medium.
5. Poly-L-lysine-coated dishes, flasks, coverslips, etc., that you want to use.
6. For mixed cortical cultures: DMEM and 10% FBS and 40 ug/ml gentamicin
On the day of dissection and plating, place the trypsin, SBTI, and G3
medium in the 37℃ incubator to equilibrate (temperature and CO2). Uncover the tissue culture plates you want under UV light to disinfect and dry.
实验步骤
1. Place the
instruments in ethanol （EtOH） and some HBSS in two petri dishes on ice.
Try to bury them without contaminating with ice. Put a 15 ml tube on ice
too. Ethanol down the microscope.
2. Sacrifice the pregnant mouse
by cervical dislocation or another acceptable method. Open the abdomen
and remove the two horns of the uterus with ethanol-sterilised scissors
into a dish of HBSS. Take out the pups which are still in their
placental sacs, and open the sacs without hurting the pups’ heads, which
are very soft. Remove the umbilical cord. With both fetuses and pups,
use sterilised scissors to cut off their heads.
3. Place the heads on the cover of the HBSS dish and ethanol them well.
4. Use the blunt forceps to immobilize the heads to the dish through
the eyes. (Don’t squeeze or push the brain!!) Use finer forceps to peel
away the scalp and then the skull. Be careful with the fetuses because
their tissues are very very soft. Make sure to get rid of all of the
soft skull, else you’ll destroy the brain when you try to remove it.
5. Using the pointed end of the spatula, separate the brain stem from
the spine and place the brain into the other dish of cold HBSS. Make
sure to keep that dish on ice. Continue with the rest of the heads.
6. Put the brains on the stage of the microscope and adjust light or
focus so you’re confortable with the dissection. Using the curved
forceps (to immobilise the cortex on the dish) and the dissecting
scalpel, take off both cortices following the circle of Willis (red
vessels). Try to use a cutting motion and go directly up and down the
circle. Put the rest of the brain away for disposal. Continue with the
rest of the brains.
7. Remove the meninges from the cortices with
fine forceps. Use one pair to stabilise the cortex, and the other to
gently lift the meninges. If you’re lucky it’ll come away in a sheet
starting at the olfactory bulb:
8. Turn the cortex around and finish
removing the meninges. The hippocampus is connected at the convex side
to the cortex, and at the concave side to more meninges. Get rid of all
of the meninges using fine forceps.
9. Use fine forceps to “cut”
the hippocampus away from the cortex at the convex side. In the end you
will have a half-circle ring.
10. Put the hippocampi away to one
side, making sure the meninges don’t contaminate them. You might prefer
to have a dish of clean HBSS on ice just for this.
11. If you want
to take the cortices for mixed cortical cultures, save the remainder of
the cortex and put into another 15 ml tube that has ice-cold HBSS on
ice. See below (step 19) for mixed cortical cultures.
12. At the
tissue culture hood, make sure that the hippocampi have settled to the
bottom of the tube and then aspirate off the HBSS. Add 1ml of warmed
trypsin per 3 brains (6 hippocampi) and vortex the tube. Put into the
incubator for 10-15 minutes.
13. Aspirate off the trypsin and add
same volume of warmed SBTI to hippocampi. Vortex well and leave at room
temperature（RT）for a couple of minutes.
14. Aspirate off the SBTI and add ~2 ml of warmed G3 medium to hippocampi.
15. Use a smoothly-fire-polished pipette to triturate. When you see no
chunks of tissue (after about 15 strokes you should see very few; you
can do maybe another 5 strokes but more than that you’ll need to strain
through a strainer-use the 70um ones behind you), stop and add enough
medium to the tube to plate. This means that for the first couple of
times you’ll need to count cells before plating, to get in your head the
approximate number of cells you can dissect. I find it helpful to
dilute the cells to the desired concentration and then put 100ul per
12mm coverslip-it saves on medium and the cells are congregated on the
coverslip.
16. Leave the plate in the incubator. After 3-5 hours of
incubation, you have to change the medium. Tilt the dish so that the
old medium runs off to the side, and aspirate that off. Careful not to
disturb the settling cells. Add the full amount of medium per well and
return to incubator.
17. After 3-5 days in culture the medium must
be half-changed to G2 medium. This is the same as G3, minus the
glutamate. To half-change, just aspirate off carefully about half of the
old medium and then replace that volume with G2.
18. Half-change the medium every 3-5 days after that with G2 until you’re ready to use them.
19. Aspirate the HBSS out of the tube containing the cortices. Add 1ml
per 3-4 brains of warm trypsin and vortex. Incubate at 37℃ for 10 min.
20. Stop the trypsinization by aspirating off the trypsin and adding
equal amount of warm medium (DMEM and FBS). Vortex. Incubate at RT for a
couple of minutes.
21. Aspirate off the medium and add 2-3 ml warm
medium to the cortices. Using first a plain pasteur pipette, and then a
fire-polished pipette, triturate until all large chunks of tissue is
broken up. This should take about 20-35 strokes.
22. Filter through a 70um mesh. Wash the tube that you triturated in with 1 ml medium and add that to the mesh.
23. If you want, count cells. I usually plate about 4 brains per T-125 flask that was precoated with PLL.
24. Change the medium in a couple of days to fresh DMEM and FBS and
gentamicin. After that change medium about every 5-7 days. The culture
should be ready in about 10 days.
