7. Remove the cell culture media in the 6-well plate that contain
the EM grids.
8. Place 0.5 μl of the resuspended cells into each well of the 6-well
plate (see Note 22).
9. Add a further 1.5 μl cell culture media to each well.
10. Incubate overnight at 37
C and 5% CO 2 in a cell culture
incubator.
11. Using a light microscope, check whether cells are well dispersed on the EM grid and a sufficient number have spread
optimally on the grid producing thin cytoplasmic regions (see
Note 23).
3.2 Sample
Vitrification
3.2.1 Preparation
of Concentrated Gold
Fiducial Marker Solution
1. Place 600 μl of 10 nm gold fiducial marker solution in two
microcentrifuge tubes (see Notes 8 and 24).
2. Centrifuge microcentrifuge tubes at 20,000 Â g for 30 min to
pellet gold beads.
3. Remove 400 μl of supernatant from each tube.
4. Resuspend the pellets in the remaining liquid and combine.
5. Store at 4
C and warm up to 37
C before use.
3.2.2 Filter Paper
and Teflon Sheet
Preparation
1. Take one piece of Teflon sheet and one piece of filter paper (see
Note 25).
2. Cut Teflon sheet to the same size as the filter paper and make a
2 cm Ø hole in the center of both.
3. Place the cut Teflon sheet and filter paper on separate pads in
the blotting device as indicated by manufacture.
3.2.3 Plunge Freezing
1. Turn heat block on and set to 37
C.
2. Switch on automatic plunge freezing device and set temperature to 37
C and humidity to 90–100%.
3. Leave both devices to reach required temperature and humidity
for 15–30 min.
4. Precool the Styrofoam cryo-dewar of the automatic plunge
freezing device by filing with liquid nitrogen. Ensure liquid
nitrogen also enters the metal pot.
5. Once the liquid nitrogen has evaporated from inside the metal
pot, top-up the liquid nitrogen surrounding the metal pot
holder (see Note 26).
6. Wearing a face shield or safety glasses and insulated gloves,
prepare the liquid ethane by focusing a jet of ethane gas against
the inside surface of the cryo-cooled metal pot of the Styrofoam cryo-dewar (see Notes 27 and 28).
7. Place 6-well plate containing EM grid on heat block.
10
Daniel Serwas and Karen M. Davies
the EM grids.
8. Place 0.5 μl of the resuspended cells into each well of the 6-well
plate (see Note 22).
9. Add a further 1.5 μl cell culture media to each well.
10. Incubate overnight at 37
C and 5% CO 2 in a cell culture
incubator.
11. Using a light microscope, check whether cells are well dispersed on the EM grid and a sufficient number have spread
optimally on the grid producing thin cytoplasmic regions (see
Note 23).
3.2 Sample
Vitrification
3.2.1 Preparation
of Concentrated Gold
Fiducial Marker Solution
1. Place 600 μl of 10 nm gold fiducial marker solution in two
microcentrifuge tubes (see Notes 8 and 24).
2. Centrifuge microcentrifuge tubes at 20,000 Â g for 30 min to
pellet gold beads.
3. Remove 400 μl of supernatant from each tube.
4. Resuspend the pellets in the remaining liquid and combine.
5. Store at 4
C and warm up to 37
C before use.
3.2.2 Filter Paper
and Teflon Sheet
Preparation
1. Take one piece of Teflon sheet and one piece of filter paper (see
Note 25).
2. Cut Teflon sheet to the same size as the filter paper and make a
2 cm Ø hole in the center of both.
3. Place the cut Teflon sheet and filter paper on separate pads in
the blotting device as indicated by manufacture.
3.2.3 Plunge Freezing
1. Turn heat block on and set to 37
C.
2. Switch on automatic plunge freezing device and set temperature to 37
C and humidity to 90–100%.
3. Leave both devices to reach required temperature and humidity
for 15–30 min.
4. Precool the Styrofoam cryo-dewar of the automatic plunge
freezing device by filing with liquid nitrogen. Ensure liquid
nitrogen also enters the metal pot.
5. Once the liquid nitrogen has evaporated from inside the metal
pot, top-up the liquid nitrogen surrounding the metal pot
holder (see Note 26).
6. Wearing a face shield or safety glasses and insulated gloves,
prepare the liquid ethane by focusing a jet of ethane gas against
the inside surface of the cryo-cooled metal pot of the Styrofoam cryo-dewar (see Notes 27 and 28).
7. Place 6-well plate containing EM grid on heat block.
10
Daniel Serwas and Karen M. Davies