around the cells to wick away while blotting from the back side
of the grid. A 200 mesh grid is the best compromise for
tomography in terms of the tradeoff between carbon stability
and imageable area.
2. Any established neuronal culture protocol can be used, with
the major difference being that EM grids are added to the
bottom of the dish. NbActiv4 has worked well in this workflow,
but a variety of growth medias have been used successfully in
other studies [10–12].
3. Whatever dish you use, it is important to test how easily you
can lift the grid from the bottom before using them. 35 mm
dishes are a good comprise in terms of minimizing the number
of cells needed to plate at the appropriate density, and having
enough room to easily get the forceps underneath the grid
when removing for plunge freezing. Six-well plates can be
used, but the approach with the forceps is from a higher
angle, making it more difficult to get under the grid. Glass
bottom dishes are recommended because the grid can be gently
pushed up against the lip at the glass/plastic interface, making
it easier to work the forceps under the grid. It is worth noting
that glass bottom dishes may occasionally leak at the seam of
the plastic and glass so it is worth checking for this during the
poly-lysine coating step, before putting the grids in the dish. To
do this, touch the bottom of the plate to a paper towel multiple
times and see if there is any persistent liquid leaking.
4. Placing a sterile circle of filter paper below the slide is helpful
for retrieving grids with forceps, in the case that some of the
grids fall off the slide into the bottom of petri dish.
5. Glow discharging grids make them hydrophilic and aid in
placing them in aqueous media. To ensure that the glow discharging protocol is working appropriately, pipette ~5 μL of
water onto a freshly glow discharged grid. On a properly discharged grid, the water will spread evenly across the entire
surface. If it is still hydrophobic, the water will ball up and
avoid spreading.
6. Make sure that the grids are well separated so the entire surface
is treated. If they are overlapping, use the fine tips of the forceps
to gently push against the side of the grids until they separate.
7. To maximize cell count, triturate until as much of the visible
particles as possible have been broken up after treating the
tissue with the dissociation media. This may take longer or
shorter than the 1 min suggested in the kit protocol. Additionally, the BrainBits protocol suggests spinning cells for 1 min,
but extending the final spin to 2 min leads to a better cell
recovery.
Cryotomography of Neurons
45
of the grid. A 200 mesh grid is the best compromise for
tomography in terms of the tradeoff between carbon stability
and imageable area.
2. Any established neuronal culture protocol can be used, with
the major difference being that EM grids are added to the
bottom of the dish. NbActiv4 has worked well in this workflow,
but a variety of growth medias have been used successfully in
other studies [10–12].
3. Whatever dish you use, it is important to test how easily you
can lift the grid from the bottom before using them. 35 mm
dishes are a good comprise in terms of minimizing the number
of cells needed to plate at the appropriate density, and having
enough room to easily get the forceps underneath the grid
when removing for plunge freezing. Six-well plates can be
used, but the approach with the forceps is from a higher
angle, making it more difficult to get under the grid. Glass
bottom dishes are recommended because the grid can be gently
pushed up against the lip at the glass/plastic interface, making
it easier to work the forceps under the grid. It is worth noting
that glass bottom dishes may occasionally leak at the seam of
the plastic and glass so it is worth checking for this during the
poly-lysine coating step, before putting the grids in the dish. To
do this, touch the bottom of the plate to a paper towel multiple
times and see if there is any persistent liquid leaking.
4. Placing a sterile circle of filter paper below the slide is helpful
for retrieving grids with forceps, in the case that some of the
grids fall off the slide into the bottom of petri dish.
5. Glow discharging grids make them hydrophilic and aid in
placing them in aqueous media. To ensure that the glow discharging protocol is working appropriately, pipette ~5 μL of
water onto a freshly glow discharged grid. On a properly discharged grid, the water will spread evenly across the entire
surface. If it is still hydrophobic, the water will ball up and
avoid spreading.
6. Make sure that the grids are well separated so the entire surface
is treated. If they are overlapping, use the fine tips of the forceps
to gently push against the side of the grids until they separate.
7. To maximize cell count, triturate until as much of the visible
particles as possible have been broken up after treating the
tissue with the dissociation media. This may take longer or
shorter than the 1 min suggested in the kit protocol. Additionally, the BrainBits protocol suggests spinning cells for 1 min,
but extending the final spin to 2 min leads to a better cell
recovery.
Cryotomography of Neurons
45