16. Sterile electroporation cuvettes, 0.2 and 0.4 cm (Bio-Rad
or BTX).
17. Electroporator (Bio-Rad Gene Pulser).
18. Tissue culture flasks (T25, T75, and T175).
19. Microwell plates (6-, 12-, and 24-well plates).
20. Falcon tubes (15 and 50 mL).
21. Plastic syringes (1, 10, and 50 mL).
22. Sterile 0.22 μm filters.
23. MicroSpin™ S-200 HR Columns (Amersham).
24. Dulbecco’s modified F-12 medium.
25. Iscove’s modified Dulbecco’s medium.
26. Opti-MEM I reduced-serum medium.
27. X-gal stock solution: 50 mM K ferricyanide, 50 mM K ferrocyanide, 1 M MgCl, 2% X-gal in DMF or DMSO.
28. X-gal staining solution: 1Â PBS, 5 mM K ferricyanide, 5 mM K
ferrocyanide, 2 mM MgCl, 1 mg/mL X-gal.
29. Moviol 4-88 containing 2.5% DABCO (1,4-diazobicyclo[2.2.2]-octane).
30. Lysis buffer: 50 mM Tris–HCl pH 7.6, 150 mM NaCl, 2 mM
EDTA, 1% (v/v) Nonidet P-40 (NP40).
31. Hybond ECL nitrocellulose filter.
32. TBST: Tris-buffered saline with 0.1% Tween 20.
33. ECL Chemiluminescence kit (Amersham).
34. Starvation medium: methionine-free MEM, 2 mM glutamine,
20 mM HEPES.
35. Chase medium: E-MEM, 2 mM glutamine, 20 mM HEPES,
150 μg/mL unlabeled methionine.
2.2 Cell Lines
The following cell lines are used for alphavirus production and
studies on expression of recombinant proteins:
1. BHK-21 cells (Baby hamster kidney) (ATCC-CCL-10).
2. CHO-K1 cells (Chinese ovary cells) (ATCC-CCL-61).
3. COS7 cells (African green monkey cells) (ATCC-CRL-1651).
4. HEK293 cells (Human embryonic kidney) (ATCC
CRL-1573).
2.3 Cell
Culture Media
1. BHK-21, CHO-K1, and HEK293 cells are cultured in a 1:1
mixture of Dulbecco’s modified F-12 medium (Gibco BRL)
and Iscove’s modified Dulbecco’s medium (Gibco BRL) supplemented with 4 mM glutamine and 10% fetal calf serum
(FCS).
Alphavirus-Based Antigen Preparation
67
or BTX).
17. Electroporator (Bio-Rad Gene Pulser).
18. Tissue culture flasks (T25, T75, and T175).
19. Microwell plates (6-, 12-, and 24-well plates).
20. Falcon tubes (15 and 50 mL).
21. Plastic syringes (1, 10, and 50 mL).
22. Sterile 0.22 μm filters.
23. MicroSpin™ S-200 HR Columns (Amersham).
24. Dulbecco’s modified F-12 medium.
25. Iscove’s modified Dulbecco’s medium.
26. Opti-MEM I reduced-serum medium.
27. X-gal stock solution: 50 mM K ferricyanide, 50 mM K ferrocyanide, 1 M MgCl, 2% X-gal in DMF or DMSO.
28. X-gal staining solution: 1Â PBS, 5 mM K ferricyanide, 5 mM K
ferrocyanide, 2 mM MgCl, 1 mg/mL X-gal.
29. Moviol 4-88 containing 2.5% DABCO (1,4-diazobicyclo[2.2.2]-octane).
30. Lysis buffer: 50 mM Tris–HCl pH 7.6, 150 mM NaCl, 2 mM
EDTA, 1% (v/v) Nonidet P-40 (NP40).
31. Hybond ECL nitrocellulose filter.
32. TBST: Tris-buffered saline with 0.1% Tween 20.
33. ECL Chemiluminescence kit (Amersham).
34. Starvation medium: methionine-free MEM, 2 mM glutamine,
20 mM HEPES.
35. Chase medium: E-MEM, 2 mM glutamine, 20 mM HEPES,
150 μg/mL unlabeled methionine.
2.2 Cell Lines
The following cell lines are used for alphavirus production and
studies on expression of recombinant proteins:
1. BHK-21 cells (Baby hamster kidney) (ATCC-CCL-10).
2. CHO-K1 cells (Chinese ovary cells) (ATCC-CCL-61).
3. COS7 cells (African green monkey cells) (ATCC-CRL-1651).
4. HEK293 cells (Human embryonic kidney) (ATCC
CRL-1573).
2.3 Cell
Culture Media
1. BHK-21, CHO-K1, and HEK293 cells are cultured in a 1:1
mixture of Dulbecco’s modified F-12 medium (Gibco BRL)
and Iscove’s modified Dulbecco’s medium (Gibco BRL) supplemented with 4 mM glutamine and 10% fetal calf serum
(FCS).
Alphavirus-Based Antigen Preparation
67