TEM grids (400 mesh); Electron Microscopy Science
(EMS), USA.
2. PELCO easiGlow™ Glow Discharge Cleaning System; Ted
Pella, Inc., USA.
3. Cryogens: liquid nitrogen (N 2 ), ethane gas.
4. EMS-002 Rapid Immersion Freezer; EMS, USA.
5. TEM grids cryo-storage box, 50 L liquid N 2 Dewar, and 2 L
liquid N 2 Dewar.
2.4 Instrumentation
of Imaging Cryo-EM
Specimen
1. Gatan cryo-transfer holder (model 910 with three specimen
cartridge) with cryo-workstation, Gatan cryo-transfer holder
(model 914) with cryo-workstation.
2. Gatan SmartSet (Model 900) Cold Stage Controller, Gatan
temperature controller (Model 1905).
3. Specimen clamping rings and loading/unloading tools.
4. JEOL-2200FS TEM/STEM at 200 kV, with cryo-pole piece,
Schottky electron source, in-column Ω energy filter.
5. Gatan Ultrascan 2k  2k CCD camera.
2.5 Software
1. 2D electron micrograph acquisition: Gatan Microscopy Suite
(GMS) V1.6.
2. 3D data reconstruction: TEMography™ package including 2D
data collection (Recorder), 3D reconstruction (Composer) and
visualization (Visualizer Kai); System in Frontier Inc., Japan.
3 Methods
3.1
Archaeosome-Based
Vaccine Formulations
1. Sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp-(1,4)-β-DGlcp-(1,1)-archaeol) glycolipids are to be dissolved in chloroform–methanol and dried under N 2 gas with mild heating to
form a thin film of lipid layer. The vacuum should be applied for
at least 2 h to ensure total removal of trace solvents.
2. Two different archaeosome formulations (antigen entrapped
and empty archaeosome) should be prepared according to the
thin-film method described as Jia et al. [7].
(a) For preparation of the entrapped Ag formulation, lipid
film is hydrated with 1.0 ml of Milli-Q water containing
ovalbumin at concentration of 10 mg/ml, and the dispersions are shaken for 2–3 h at 40–50
C until completely
suspended. Next a brief sonication is applied at 40
C in an
Ultrasonic water bath until a desired particle size
(~100 nm) is obtained. Unentrapped antigen is removed
by ultracentrifugation (223,000 Â g for 1.5 h at 4
C).
502
Hui Qian et al.
(EMS), USA.
2. PELCO easiGlow™ Glow Discharge Cleaning System; Ted
Pella, Inc., USA.
3. Cryogens: liquid nitrogen (N 2 ), ethane gas.
4. EMS-002 Rapid Immersion Freezer; EMS, USA.
5. TEM grids cryo-storage box, 50 L liquid N 2 Dewar, and 2 L
liquid N 2 Dewar.
2.4 Instrumentation
of Imaging Cryo-EM
Specimen
1. Gatan cryo-transfer holder (model 910 with three specimen
cartridge) with cryo-workstation, Gatan cryo-transfer holder
(model 914) with cryo-workstation.
2. Gatan SmartSet (Model 900) Cold Stage Controller, Gatan
temperature controller (Model 1905).
3. Specimen clamping rings and loading/unloading tools.
4. JEOL-2200FS TEM/STEM at 200 kV, with cryo-pole piece,
Schottky electron source, in-column Ω energy filter.
5. Gatan Ultrascan 2k  2k CCD camera.
2.5 Software
1. 2D electron micrograph acquisition: Gatan Microscopy Suite
(GMS) V1.6.
2. 3D data reconstruction: TEMography™ package including 2D
data collection (Recorder), 3D reconstruction (Composer) and
visualization (Visualizer Kai); System in Frontier Inc., Japan.
3 Methods
3.1
Archaeosome-Based
Vaccine Formulations
1. Sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp-(1,4)-β-DGlcp-(1,1)-archaeol) glycolipids are to be dissolved in chloroform–methanol and dried under N 2 gas with mild heating to
form a thin film of lipid layer. The vacuum should be applied for
at least 2 h to ensure total removal of trace solvents.
2. Two different archaeosome formulations (antigen entrapped
and empty archaeosome) should be prepared according to the
thin-film method described as Jia et al. [7].
(a) For preparation of the entrapped Ag formulation, lipid
film is hydrated with 1.0 ml of Milli-Q water containing
ovalbumin at concentration of 10 mg/ml, and the dispersions are shaken for 2–3 h at 40–50
C until completely
suspended. Next a brief sonication is applied at 40
C in an
Ultrasonic water bath until a desired particle size
(~100 nm) is obtained. Unentrapped antigen is removed
by ultracentrifugation (223,000 Â g for 1.5 h at 4
C).
502
Hui Qian et al.