3.1.2 Cell Seeding
1. Take 10 cm plastic cell culture dish where cells are grown to
75–90% confluency.
2. Wash cells with PBS without calcium and magnesium.
3. Replace PBS with 0.05% Trypsin-EDTA solution (3 μl) and
incubate for 2 min at 37
C to detach cells.
4. Inactive Trypsin-EDTA by adding 7 μl of cell culture media and
transfer the entire volume to a 15 μl falcon tube.
5. Harvest cells by centrifugation (2 min, RT, ~180 Â g).
6. Remove supernatant and resuspend in 9 μl cell culture media.
a
Placing grid in well
Tweezer
EM grid
Well containing
cell culture
medium
Incubate grids in 6-well plate with
cell culture medium
(overnight, 37°C, 5% CO )
Renew medium, seed cells &
incubate (overnight, 37°C, 5% CO )
Seeding cells
b
2
2
Sample vitrification
c
Tweezer holding
an EM grid
Teflon sheet facing
sample side
Filter paper for
backside blotting
Metal cup with
liquid ethane
Styrofoam dewar
with liquid nitrogen
Fig. 1 EM grid handling and sample preparation outline. (a) Schematic showing how to hold a grid and
submerge it in cell culture media. EM grid should be held with the outer metal ring only and inserted into the
media orientated 90
to liquid surface. Once submerged, the grid can be tilted and laid flat on the well floor
with carbon side up. (b) Workflow of EM grid seeding. Left: EM grids are incubated overnight, carbon side up in
6-well plates containing 2 ml of cell culture medium. Right: After incubation, cell culture medium is renewed,
and cells added. Cells are allowed to settle on grids overnight before plunge freezing. (c) Seeded EM grids are
removed from cell culture plate and placed in freezing device with cells facing Teflon sheet. After backside
blotting, grids are plunged into liquid ethane and transferred to storage device under liquid nitrogen
Cryo-ET of Cellular Structures
9
1. Take 10 cm plastic cell culture dish where cells are grown to
75–90% confluency.
2. Wash cells with PBS without calcium and magnesium.
3. Replace PBS with 0.05% Trypsin-EDTA solution (3 μl) and
incubate for 2 min at 37
C to detach cells.
4. Inactive Trypsin-EDTA by adding 7 μl of cell culture media and
transfer the entire volume to a 15 μl falcon tube.
5. Harvest cells by centrifugation (2 min, RT, ~180 Â g).
6. Remove supernatant and resuspend in 9 μl cell culture media.
a
Placing grid in well
Tweezer
EM grid
Well containing
cell culture
medium
Incubate grids in 6-well plate with
cell culture medium
(overnight, 37°C, 5% CO )
Renew medium, seed cells &
incubate (overnight, 37°C, 5% CO )
Seeding cells
b
2
2
Sample vitrification
c
Tweezer holding
an EM grid
Teflon sheet facing
sample side
Filter paper for
backside blotting
Metal cup with
liquid ethane
Styrofoam dewar
with liquid nitrogen
Fig. 1 EM grid handling and sample preparation outline. (a) Schematic showing how to hold a grid and
submerge it in cell culture media. EM grid should be held with the outer metal ring only and inserted into the
media orientated 90
to liquid surface. Once submerged, the grid can be tilted and laid flat on the well floor
with carbon side up. (b) Workflow of EM grid seeding. Left: EM grids are incubated overnight, carbon side up in
6-well plates containing 2 ml of cell culture medium. Right: After incubation, cell culture medium is renewed,
and cells added. Cells are allowed to settle on grids overnight before plunge freezing. (c) Seeded EM grids are
removed from cell culture plate and placed in freezing device with cells facing Teflon sheet. After backside
blotting, grids are plunged into liquid ethane and transferred to storage device under liquid nitrogen
Cryo-ET of Cellular Structures
9