In this book chapter, we aim to guide the reader through the
principle steps required to generate and analyze a cryo-electron
tomogram of mammalian cells. To achieve this, we provide a stepby-step protocol for the initial sample preparation and suggest a
general method for data collection, tomographic reconstruction,
and analyses. We hope that this chapter will encourage more people
to pursue cryo-ET.
2 Materials
2.1 Growing Cells
on Grids
2.1.1 Grid Preparation
1. Gold EM grids, 200 mesh with holey carbon support films (see
Note 1).
2. Tweezers, Type 5 (see Note 2).
3. Two 10 cm Ø Pyrex
® petri dishes.
4. Acetone (!99.5%).
5. Fixed speed rocker with platform to accommodate a 10 cm Ø
petri dish.
6. Filter paper (Whatman No. 1, 90 mm Ø).
7. EM Grid storage box (see Note 3).
8. Glow discharging device.
9. 70% ethanol solution in H 2 O: For 1000 μl, mix 700 μl anhydrous ethanol with dH 2 O.
Carbon Coating (Optional)
1. Muscovite Mica sheets, V-1 Quality (see Note 4).
2. Carbon rods (see Note 5).
3. Carbon evaporator.
4. Smith Grid Coating Trough.
Extracellular Matrix Coating
(Optional)
1. Sterile solutions of extracellular matrix components (e.g. polyL-lysine).
2.1.2 Cell Seeding
1. Biosafety cabinet.
2. 6-Well cell culture plate (sterile).
3. Cell culture media: For SK-MEL-2 cells, use Gibco™
DMEM/F12 supplemented with 10% fetal bovine serum (premium grade, VWR Seradigm Life Science) and 1% Gibco™
Penicillin/Streptomycin (see Note 6).
4. 10 cm Ø plastic cell culture dish with SK-MEL-2 cells (ATCC
®
HTB-68™) grown to 75–90% confluency (see Note 7).
5. Cell culture incubator set to 37
C and 5% CO 2 .
6. Gibco™ Dulbecco’s Phosphate Buffered Saline (PBS) without
Calcium and Magnesium (sterile).
6
Daniel Serwas and Karen M. Davies
principle steps required to generate and analyze a cryo-electron
tomogram of mammalian cells. To achieve this, we provide a stepby-step protocol for the initial sample preparation and suggest a
general method for data collection, tomographic reconstruction,
and analyses. We hope that this chapter will encourage more people
to pursue cryo-ET.
2 Materials
2.1 Growing Cells
on Grids
2.1.1 Grid Preparation
1. Gold EM grids, 200 mesh with holey carbon support films (see
Note 1).
2. Tweezers, Type 5 (see Note 2).
3. Two 10 cm Ø Pyrex
® petri dishes.
4. Acetone (!99.5%).
5. Fixed speed rocker with platform to accommodate a 10 cm Ø
petri dish.
6. Filter paper (Whatman No. 1, 90 mm Ø).
7. EM Grid storage box (see Note 3).
8. Glow discharging device.
9. 70% ethanol solution in H 2 O: For 1000 μl, mix 700 μl anhydrous ethanol with dH 2 O.
Carbon Coating (Optional)
1. Muscovite Mica sheets, V-1 Quality (see Note 4).
2. Carbon rods (see Note 5).
3. Carbon evaporator.
4. Smith Grid Coating Trough.
Extracellular Matrix Coating
(Optional)
1. Sterile solutions of extracellular matrix components (e.g. polyL-lysine).
2.1.2 Cell Seeding
1. Biosafety cabinet.
2. 6-Well cell culture plate (sterile).
3. Cell culture media: For SK-MEL-2 cells, use Gibco™
DMEM/F12 supplemented with 10% fetal bovine serum (premium grade, VWR Seradigm Life Science) and 1% Gibco™
Penicillin/Streptomycin (see Note 6).
4. 10 cm Ø plastic cell culture dish with SK-MEL-2 cells (ATCC
®
HTB-68™) grown to 75–90% confluency (see Note 7).
5. Cell culture incubator set to 37
C and 5% CO 2 .
6. Gibco™ Dulbecco’s Phosphate Buffered Saline (PBS) without
Calcium and Magnesium (sterile).
6
Daniel Serwas and Karen M. Davies