3.3 Neuronal Culture
1. Obtain E18 rat hippocampal neurons. We use a kit from BrainBits, but any protocol for obtaining primary neurons should
suffice. The kit can be stored at 4
C for up to 3–4 days, but
there may be a drop-off in the total number of living cells
acquired from the tissue.
2. If using the kit, follow the protocol outlined below (or follow
the kit protocol):
(a) Make the cell dissociation solution by dissolving the
provided 6 mg of Papain in 3 mL Hibernate E without
Calcium (HE-Ca) in a 30
C water bath for 10 min.
(b) Using a Pasteur pipette, place hippocampi in the cell
dissociation solution and incubate in a 30
C water bath
for 10 min.
(c) Use the Pasteur pipette to return the hippocampi to its
original vial/media.
(d) Triturate tissue for ~1 min to dissociate cells into a single
cell suspension (see Note 7).
(e) Let heavy debris settle to the bottom of the tube for at
least 1 min, before carefully transferring the cell suspension into a sterile 15 mL conical (leaving behind any
debris).
(f) Spin single cell suspension at 256 Â g for 2 min (see Note
7).
(g) Discard supernatant and resuspend the pellet in 1 mL of
growth media.
3. Count cells in a hemocytometer using trypan blue exclusion.
4. Dilute cell in growth media to desired plating density, assuming
2 mL of media per 35 mm dish.
5. Aspirate water or media from 35 mm dishes and plate cells at
20-40 k cells/cm
2 over the grid-containing plates. Plating
192 k, 288 k, or 384 k cells/dish in 2 mL of media will result
in 20 k, 30 k, and 40 k cells/cm
2 , respectively.
6. If grids move during plating, separate them by gently pushing
them away from one another with forceps.
7. Check cell distribution on a light microscope to ensure that
cells are monodispersed.
8. Feed cells every 4 days by exchanging half of the media (1 mL)
with fresh media, out to the number of days in vitro desired (see
Fig. 2).
Cryotomography of Neurons
29
1. Obtain E18 rat hippocampal neurons. We use a kit from BrainBits, but any protocol for obtaining primary neurons should
suffice. The kit can be stored at 4
C for up to 3–4 days, but
there may be a drop-off in the total number of living cells
acquired from the tissue.
2. If using the kit, follow the protocol outlined below (or follow
the kit protocol):
(a) Make the cell dissociation solution by dissolving the
provided 6 mg of Papain in 3 mL Hibernate E without
Calcium (HE-Ca) in a 30
C water bath for 10 min.
(b) Using a Pasteur pipette, place hippocampi in the cell
dissociation solution and incubate in a 30
C water bath
for 10 min.
(c) Use the Pasteur pipette to return the hippocampi to its
original vial/media.
(d) Triturate tissue for ~1 min to dissociate cells into a single
cell suspension (see Note 7).
(e) Let heavy debris settle to the bottom of the tube for at
least 1 min, before carefully transferring the cell suspension into a sterile 15 mL conical (leaving behind any
debris).
(f) Spin single cell suspension at 256 Â g for 2 min (see Note
7).
(g) Discard supernatant and resuspend the pellet in 1 mL of
growth media.
3. Count cells in a hemocytometer using trypan blue exclusion.
4. Dilute cell in growth media to desired plating density, assuming
2 mL of media per 35 mm dish.
5. Aspirate water or media from 35 mm dishes and plate cells at
20-40 k cells/cm
2 over the grid-containing plates. Plating
192 k, 288 k, or 384 k cells/dish in 2 mL of media will result
in 20 k, 30 k, and 40 k cells/cm
2 , respectively.
6. If grids move during plating, separate them by gently pushing
them away from one another with forceps.
7. Check cell distribution on a light microscope to ensure that
cells are monodispersed.
8. Feed cells every 4 days by exchanging half of the media (1 mL)
with fresh media, out to the number of days in vitro desired (see
Fig. 2).
Cryotomography of Neurons
29