2.2.1 Preparing Cells
Sample preparation depends on the cell type. Here we give an
example for U2OS cells.
U2OS cells should be grown in a T75 flask containing McCoy’s
5a Medium Modified (see Note 1). Typically, U2OS cells are passaged every 2 days and new media replaced every day. At all times,
the cells are incubated at 37
C with 5% CO 2 . The U2OS cells are
grown to 80% confluency in a T75 flask before being detached by
trypsin and seeded on EM grids. Usually, 1 mL of trypsin is added
to the flask and left to incubate at 37
C for 2 min. Next, 6 mL of
media is added to the flask to neutralize the trypsin (see Note 2). To
remove clumps and achieve a single-cell suspension, the cells are
pipetted up and down 20 times using a 10 mL pipette. A cell
strainer can also be used to obtain a uniform single-cell suspension
from mammalian cells. The suspended cells are then transferred to a
10 mL Falcon tube.
2.2.2 Preparing EM Grids
for Tissue Culture
1. London finder grids with Quantifoil holey carbon are UV
treated for 1 h before plating experiment (see Note 3). Specific
extracellular matrix proteins (ECMs) can be used to improve
attachment and spreading of various cells. The most commonly
used ECMs are collagen, fibronectin, poly-L-lysine, and laminin. For U2OS cells, we use collagen. Several drops of human
collagen diluted ten times in H 2 O are pipetted onto parafilm.
2. A UV-treated grid is then placed carbon-side down on the drop
for 2 min. The grid is then flipped so that the grid sinks to the
bottom of the drop with the carbon-side now facing up (see
Note 4). The grid is left in the drop for 20 min.
3. Once the grid has incubated in the drop, it is picked up with
tweezers and the excess collagen solution on the grid is blotted
away with Whatman 40 blotting paper and the grid is left
to dry.
2.2.3 Growing
Mammalian Cells on Grids
1. Glass slides are placed in a 10 mL petri dish and UV treated for
20 min. 20 mLs of media is then added to the petri dish to
cover the glass slides. Alternatively, when smaller volumes of
samples are required a 35 mm imaging dish with coverslip
bottom can be used.
2. The pre-coated EM grids are then submerged into the media of
the petri dish with the carbon side of the grid facing upwards
and placed on the glass slide.
3. The detached cells are then added to the petri dish. The optimal density for growing cells on R2/2 grids is 1 or 2 cells per
grid square. If there are more than three cells per grid square,
there will be little room for the cells to spread and grow flat.
Plating cells on grids so that roughly one or two cells are
present in most of the grid squares can be achieved by
Methods in Cryo-Electron Tomography
87
Sample preparation depends on the cell type. Here we give an
example for U2OS cells.
U2OS cells should be grown in a T75 flask containing McCoy’s
5a Medium Modified (see Note 1). Typically, U2OS cells are passaged every 2 days and new media replaced every day. At all times,
the cells are incubated at 37
C with 5% CO 2 . The U2OS cells are
grown to 80% confluency in a T75 flask before being detached by
trypsin and seeded on EM grids. Usually, 1 mL of trypsin is added
to the flask and left to incubate at 37
C for 2 min. Next, 6 mL of
media is added to the flask to neutralize the trypsin (see Note 2). To
remove clumps and achieve a single-cell suspension, the cells are
pipetted up and down 20 times using a 10 mL pipette. A cell
strainer can also be used to obtain a uniform single-cell suspension
from mammalian cells. The suspended cells are then transferred to a
10 mL Falcon tube.
2.2.2 Preparing EM Grids
for Tissue Culture
1. London finder grids with Quantifoil holey carbon are UV
treated for 1 h before plating experiment (see Note 3). Specific
extracellular matrix proteins (ECMs) can be used to improve
attachment and spreading of various cells. The most commonly
used ECMs are collagen, fibronectin, poly-L-lysine, and laminin. For U2OS cells, we use collagen. Several drops of human
collagen diluted ten times in H 2 O are pipetted onto parafilm.
2. A UV-treated grid is then placed carbon-side down on the drop
for 2 min. The grid is then flipped so that the grid sinks to the
bottom of the drop with the carbon-side now facing up (see
Note 4). The grid is left in the drop for 20 min.
3. Once the grid has incubated in the drop, it is picked up with
tweezers and the excess collagen solution on the grid is blotted
away with Whatman 40 blotting paper and the grid is left
to dry.
2.2.3 Growing
Mammalian Cells on Grids
1. Glass slides are placed in a 10 mL petri dish and UV treated for
20 min. 20 mLs of media is then added to the petri dish to
cover the glass slides. Alternatively, when smaller volumes of
samples are required a 35 mm imaging dish with coverslip
bottom can be used.
2. The pre-coated EM grids are then submerged into the media of
the petri dish with the carbon side of the grid facing upwards
and placed on the glass slide.
3. The detached cells are then added to the petri dish. The optimal density for growing cells on R2/2 grids is 1 or 2 cells per
grid square. If there are more than three cells per grid square,
there will be little room for the cells to spread and grow flat.
Plating cells on grids so that roughly one or two cells are
present in most of the grid squares can be achieved by
Methods in Cryo-Electron Tomography
87