Nvidia-based video card (4 GB or higher; AMD cards can be
used for Windows or Mac) and 16 GB RAM See also: https://
bio3d.colorado.edu/imod/, Note 13).
2. Installation of latest stable IMOD version ([45], https://
bio3d.colorado.edu/imod/).
3 Methods
3.1 Growing Cells
on Grids
All work involving mammalian cells is performed in a biosafety
cabinet unless otherwise stated. Grids should always be handled
with fine forceps.
3.1.1 Grid Preparation
1. When handling grids, make sure to touch only the outer metal
part of the grid with the tweezers to prevent damage to the
supporting film (Fig. 1a).
2. In a fume hood, place the required amount of holey carbon
grids in a 10 cm Ø Pyrex
® petri dish containing acetone and
gently agitate on a rocker for 15–30 min at RT to remove
residual plastic backing (see Notes 14 and 15).
3. Dry grids on a filter paper and store in grid box.
4. If required, apply a thin carbon film (~8 nm) to the cleaned EM
grids (see Notes 15–18).
5. Plasma clean grids in a glow discharging device to make the
surface hydrophilic.
6. Sterilize grids by placing grids in a 10 cm Ø Pyrex
® petri dish
with 70% ethanol in H 2 O and gently agitate on a rocker for
15–30 min at RT.
7. Move to a biosafety cabinet.
8. If required: coat grids with extracellular matrix to improve cell
adhesion. Remove grid from ethanol and drain excess ethanol
by touching grid edge to surface of ethanol. (Do not
completely dry grid). Place grid into 6-well plate containing
~1.5 μl of extracellular matrix protein solution. Ensure carbon
side faces up (see Note 19). Incubate at 37
C in cell culture
incubator for 1–2 h.
9. After coating or if no coating is required, pick up grids and
drain off excess liquid by touching the edge of grid to the
surface of the solution.
10. Place EM grid in a 6-well plate where each well contains 2 μl of
cell culture media. Place only one grid per well with carbon side
up (see Note 20, Fig. 1b).
11. Incubate overnight at 37
C and 5% CO 2 in a cell culture
incubator (see Note 21).
8
Daniel Serwas and Karen M. Davies
used for Windows or Mac) and 16 GB RAM See also: https://
bio3d.colorado.edu/imod/, Note 13).
2. Installation of latest stable IMOD version ([45], https://
bio3d.colorado.edu/imod/).
3 Methods
3.1 Growing Cells
on Grids
All work involving mammalian cells is performed in a biosafety
cabinet unless otherwise stated. Grids should always be handled
with fine forceps.
3.1.1 Grid Preparation
1. When handling grids, make sure to touch only the outer metal
part of the grid with the tweezers to prevent damage to the
supporting film (Fig. 1a).
2. In a fume hood, place the required amount of holey carbon
grids in a 10 cm Ø Pyrex
® petri dish containing acetone and
gently agitate on a rocker for 15–30 min at RT to remove
residual plastic backing (see Notes 14 and 15).
3. Dry grids on a filter paper and store in grid box.
4. If required, apply a thin carbon film (~8 nm) to the cleaned EM
grids (see Notes 15–18).
5. Plasma clean grids in a glow discharging device to make the
surface hydrophilic.
6. Sterilize grids by placing grids in a 10 cm Ø Pyrex
® petri dish
with 70% ethanol in H 2 O and gently agitate on a rocker for
15–30 min at RT.
7. Move to a biosafety cabinet.
8. If required: coat grids with extracellular matrix to improve cell
adhesion. Remove grid from ethanol and drain excess ethanol
by touching grid edge to surface of ethanol. (Do not
completely dry grid). Place grid into 6-well plate containing
~1.5 μl of extracellular matrix protein solution. Ensure carbon
side faces up (see Note 19). Incubate at 37
C in cell culture
incubator for 1–2 h.
9. After coating or if no coating is required, pick up grids and
drain off excess liquid by touching the edge of grid to the
surface of the solution.
10. Place EM grid in a 6-well plate where each well contains 2 μl of
cell culture media. Place only one grid per well with carbon side
up (see Note 20, Fig. 1b).
11. Incubate overnight at 37
C and 5% CO 2 in a cell culture
incubator (see Note 21).
8
Daniel Serwas and Karen M. Davies