7. 0.05% Trypsin-EDTA solution (Gibco™).
8. Sterile plastic pipettes (10 and 5 μl).
9. Pipette controller (‘Pipetboy’).
10. Conical falcon tube (15 μl).
11. Centrifuge (for pelleting cells at ~180 Â g; should be able to
accommodate 15 μl conical falcon tubes).
12. Inverted light microscope equipped with a 4Â and 10Â Objective to monitor cell growth control.
2.2 Sample
Vitrification
1. 10 nm Gold Fiducial Markers (see Note 8).
2. Two microcentrifuge tubes (1.5 μl).
3. Micropipettes (1000, 200 and 20 μl) and pipette tips.
4. Benchtop centrifuge which handles microcentrifuge tubes.
5. Teflon sheet (see Note 9).
6. Scissors.
7. Filter paper (Whatman No. 1, 55 mm Ø).
8. Heat block set to 37
C (should be able to accommodate 6-well
plate).
9. Heat block set to 37
C (should be able to accommodate 1.5 μl
microcentrifuge tubes).
10. Automatic plunge freezing device (see Note 10).
11. Liquid nitrogen.
12. Ethane gas (99.999% pure).
13. Conical falcon tube with string (50 μl, for storage of cryo-grid
boxes with vitrified samples, see Note 11).
14. Liquid nitrogen dewars (4 L, smaller ones for sample transport
in 50 μl conical falcon tube).
15. Cryo-EM grid boxes (see Note 12).
16. Gripper tool for cryo-grid boxes (see Note 12).
17. Liquid nitrogen sample storage dewar.
2.3 Data Collection
1. 300 kV cryo-TEM (e.g. Krios G3i, Thermo Fisher Scientific,
previously FEI) equipped with energy filter (e.g. Bioquantum,
Gatan) and direct electron detecting device (e.g. K2). Optional
equipment: Phase plate (e.g. Volta Phase Plate).
2. Latest version of SerialEM software installed on computer
controlling microscope camera ([44], http://bio3d.colorado.
edu/SerialEM/).
2.4 Data Processing
1. Workstation(s) (Linux, Windows or Mac operating systems can
be used) for data processing. Minimal configuration: Desktop
computer with modern Intel or AMD processor, modern
Cryo-ET of Cellular Structures
7
8. Sterile plastic pipettes (10 and 5 μl).
9. Pipette controller (‘Pipetboy’).
10. Conical falcon tube (15 μl).
11. Centrifuge (for pelleting cells at ~180 Â g; should be able to
accommodate 15 μl conical falcon tubes).
12. Inverted light microscope equipped with a 4Â and 10Â Objective to monitor cell growth control.
2.2 Sample
Vitrification
1. 10 nm Gold Fiducial Markers (see Note 8).
2. Two microcentrifuge tubes (1.5 μl).
3. Micropipettes (1000, 200 and 20 μl) and pipette tips.
4. Benchtop centrifuge which handles microcentrifuge tubes.
5. Teflon sheet (see Note 9).
6. Scissors.
7. Filter paper (Whatman No. 1, 55 mm Ø).
8. Heat block set to 37
C (should be able to accommodate 6-well
plate).
9. Heat block set to 37
C (should be able to accommodate 1.5 μl
microcentrifuge tubes).
10. Automatic plunge freezing device (see Note 10).
11. Liquid nitrogen.
12. Ethane gas (99.999% pure).
13. Conical falcon tube with string (50 μl, for storage of cryo-grid
boxes with vitrified samples, see Note 11).
14. Liquid nitrogen dewars (4 L, smaller ones for sample transport
in 50 μl conical falcon tube).
15. Cryo-EM grid boxes (see Note 12).
16. Gripper tool for cryo-grid boxes (see Note 12).
17. Liquid nitrogen sample storage dewar.
2.3 Data Collection
1. 300 kV cryo-TEM (e.g. Krios G3i, Thermo Fisher Scientific,
previously FEI) equipped with energy filter (e.g. Bioquantum,
Gatan) and direct electron detecting device (e.g. K2). Optional
equipment: Phase plate (e.g. Volta Phase Plate).
2. Latest version of SerialEM software installed on computer
controlling microscope camera ([44], http://bio3d.colorado.
edu/SerialEM/).
2.4 Data Processing
1. Workstation(s) (Linux, Windows or Mac operating systems can
be used) for data processing. Minimal configuration: Desktop
computer with modern Intel or AMD processor, modern
Cryo-ET of Cellular Structures
7