Neutralization Assay Protocol

Procedure for Cell Viability: MTT - Thiazolyl Blue Tetrazolium Bromide

1. Make a 5 mg/mL MTT solution in sterile autoclaved PBS (5 mg/mL). Store at 4°C protected from light.
2. Add 25 µL MTT per well without removing medium from plate. Incubate 2 hours at 37°C.
3. Add 100 µL extraction buffer (12.5% SDS, 45% dimethylformamide, pH 4.7) and allow to incubate overnight at 37°C (to dissolve formazan-protein complexes in living/enzymatically active cells).
4. Read absorbance at 570 nm.

Procedure for Cell Viability: Amido Blue-black

1. Stain cells with 0.05% amido blue-black solution.
2. Fix cells with 10% formalin solution in acetic acid buffer.
3. Elute Amido blue-black stain in 0.15 mL of 0.05 M NaOH solution per well.
4. Read ABS at 620 nm.

References

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• T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. (1983) Dec 16;65(1-2):55-63.
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• “Guidelines for Plaque Reduction Neutralization Testing of Human antibodies to Dengue virus” Initiative for Vaccine Research of the Department of Immunization, Vaccines and Biologicals. World Health Organization. Department of Immunization, Vaccines and Biologicals.