7. Coating buffer: 0.1 M NaHCO 3 , pH 9.6.
8. Biotinylated anti-mouse immunoglobulin of interest (Abcam).
9. HRP-conjugated streptavidin (BD Bioscience).
10. Blocking buffer: 1% bovine serum albumin (BSA) in Phosphate
buffered saline (PBS).
11. Washing buffer: PBS + 0.05% Tween20 (Sigma-Aldrich).
12. TMB substrate kit (KPL, SeraCare).
13. Stop solution: 1 M phosphoric acid (Sigma-Aldrich).
14. Plate washer (NUNC).
15. Plate reader (OD 450nm ).
2.5 Plaque
Reduction Assay
1. Class II biosafety cabinet.
2. Water bath.
3. CO 2 incubator (Thermo Scientific).
4. Vero cells (ATCC CCL-81).
5. Assay virus (JEV Nakayama strain).
6. Sterile serological pipettes (Greiner Bio-One).
7. Dulbecco’s Modified Eagle Medium (1Â DMEM) (Gibco,
ThermoFisher).
8. Double strength Minimum Essential Medium (2Â MEM)
(Gibco, ThermoFisher).
9. Fetal calf serum (FCS) (Gibco, ThermoFisher).
10. PBS (Sigma-Aldrich).
11. 0.25% trypsin–EDTA (Sigma-Aldrich).
12. 100Â penicillin–streptomycin (10,000 U/mL) (Gibco,
ThermoFisher).
13. 100Â GlutaMAX Supplement (Gibco, ThermoFisher).
14. 0.22 μm Disk filter unit and 50 mL syringe.
15. Variable Adjustable Volume Pipettes.
16. Hemocytometer.
17. Inverted microscope (Olympus).
18. Avicel RC-591 (FMC BioPolymer).
19. Formalin (Sigma-Aldrich).
20. Methylene blue (Sigma-Aldrich).
2.6 ELISPOT Assay
1. Laminar flow hood.
2. 70 μm Cell strainer (Falcon, Corning).
3. 2 mL disposable syringe and 25-G needle (BD).
4. RPMI1640 medium (Gibco, ThermoFisher).
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Lei Li et al.
8. Biotinylated anti-mouse immunoglobulin of interest (Abcam).
9. HRP-conjugated streptavidin (BD Bioscience).
10. Blocking buffer: 1% bovine serum albumin (BSA) in Phosphate
buffered saline (PBS).
11. Washing buffer: PBS + 0.05% Tween20 (Sigma-Aldrich).
12. TMB substrate kit (KPL, SeraCare).
13. Stop solution: 1 M phosphoric acid (Sigma-Aldrich).
14. Plate washer (NUNC).
15. Plate reader (OD 450nm ).
2.5 Plaque
Reduction Assay
1. Class II biosafety cabinet.
2. Water bath.
3. CO 2 incubator (Thermo Scientific).
4. Vero cells (ATCC CCL-81).
5. Assay virus (JEV Nakayama strain).
6. Sterile serological pipettes (Greiner Bio-One).
7. Dulbecco’s Modified Eagle Medium (1Â DMEM) (Gibco,
ThermoFisher).
8. Double strength Minimum Essential Medium (2Â MEM)
(Gibco, ThermoFisher).
9. Fetal calf serum (FCS) (Gibco, ThermoFisher).
10. PBS (Sigma-Aldrich).
11. 0.25% trypsin–EDTA (Sigma-Aldrich).
12. 100Â penicillin–streptomycin (10,000 U/mL) (Gibco,
ThermoFisher).
13. 100Â GlutaMAX Supplement (Gibco, ThermoFisher).
14. 0.22 μm Disk filter unit and 50 mL syringe.
15. Variable Adjustable Volume Pipettes.
16. Hemocytometer.
17. Inverted microscope (Olympus).
18. Avicel RC-591 (FMC BioPolymer).
19. Formalin (Sigma-Aldrich).
20. Methylene blue (Sigma-Aldrich).
2.6 ELISPOT Assay
1. Laminar flow hood.
2. 70 μm Cell strainer (Falcon, Corning).
3. 2 mL disposable syringe and 25-G needle (BD).
4. RPMI1640 medium (Gibco, ThermoFisher).
410
Lei Li et al.