9. LB medium.
10. LB agar plates.
11. Ni-NTA Superflow resin (Qiagen).
12. Sodium phosphate buffer.
13. 150 mM sodium chloride buffer.
14. Imidazole.
15. Complete EDTA-free protease inhibitor cocktail tablets
(Roche Diagnostics).
16. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), sample buffer, and running buffer (Invitrogen).
17. β-Mercaptoethanol.
18. SDS-PAGE electrophoresis chamber.
2.2 Vaccination,
Immune Responses,
and Dual Challenge
2.2.1 Immunization
of Mice and Serum
Collection
1. BALB/c mice (6–8 weeks) from Harlan Laboratories
(Indianapolis, IN), A/J mice (6–8 weeks), and
CD4-knockout (KO) and CD8-KO C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME).
2. Antigen EFn-3ÂM2e-HA2.
3. Recombinant anthrax protective antigen (BEI Resources,
Manassas, VA).
4. 5-mm lancets to bleed mice using the submandibular method.
5. Ketamine and xylazine.
2.3 T Cell
and Antibody
Responses
and Protection
2.3.1 ELISA
1. Mouse sera of immunized individuals.
2. Phosphate-buffered saline (PBS), pH 7.2.
3. Tween 20 detergent.
4. Immuno Clear standard module cx320.
5. Bovine serum albumin.
6. Anti-mouse IgG-Fc, IgG1, and IgG2a-phosphatase.
7. p-Nitrophenylphosphate phosphatase substrate system (KPL,
Gaithersburg, MD).
8. ImmunoWash™ 1575 microplate washer (Bio-Rad,
Hercules, CA).
9. PowereWave XS2 plate reader with GEN5 software (BioTek,
Winooski, VT).
2.3.2 Influenza Virus
Microneutralization Assay
1. H1N1 influenza virus A/PR/8/34 (PR8, from VR-95;
ATCC, Manassas, VA).
2. Madin–Darby canine kidney (MDCK) cells.
3. 1% agarose in medium (minimum essential medium, 0.2% BSA,
1 μg/ml TPCK-treated trypsin).
4. Ethanol.
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Diana Diaz-Are ´ valo et al.
10. LB agar plates.
11. Ni-NTA Superflow resin (Qiagen).
12. Sodium phosphate buffer.
13. 150 mM sodium chloride buffer.
14. Imidazole.
15. Complete EDTA-free protease inhibitor cocktail tablets
(Roche Diagnostics).
16. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), sample buffer, and running buffer (Invitrogen).
17. β-Mercaptoethanol.
18. SDS-PAGE electrophoresis chamber.
2.2 Vaccination,
Immune Responses,
and Dual Challenge
2.2.1 Immunization
of Mice and Serum
Collection
1. BALB/c mice (6–8 weeks) from Harlan Laboratories
(Indianapolis, IN), A/J mice (6–8 weeks), and
CD4-knockout (KO) and CD8-KO C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME).
2. Antigen EFn-3ÂM2e-HA2.
3. Recombinant anthrax protective antigen (BEI Resources,
Manassas, VA).
4. 5-mm lancets to bleed mice using the submandibular method.
5. Ketamine and xylazine.
2.3 T Cell
and Antibody
Responses
and Protection
2.3.1 ELISA
1. Mouse sera of immunized individuals.
2. Phosphate-buffered saline (PBS), pH 7.2.
3. Tween 20 detergent.
4. Immuno Clear standard module cx320.
5. Bovine serum albumin.
6. Anti-mouse IgG-Fc, IgG1, and IgG2a-phosphatase.
7. p-Nitrophenylphosphate phosphatase substrate system (KPL,
Gaithersburg, MD).
8. ImmunoWash™ 1575 microplate washer (Bio-Rad,
Hercules, CA).
9. PowereWave XS2 plate reader with GEN5 software (BioTek,
Winooski, VT).
2.3.2 Influenza Virus
Microneutralization Assay
1. H1N1 influenza virus A/PR/8/34 (PR8, from VR-95;
ATCC, Manassas, VA).
2. Madin–Darby canine kidney (MDCK) cells.
3. 1% agarose in medium (minimum essential medium, 0.2% BSA,
1 μg/ml TPCK-treated trypsin).
4. Ethanol.
426
Diana Diaz-Are ´ valo et al.