Materials and Reagents

Monkey kidney cell line CV-1 cells
MDCK-propagated Influenza B viruses (B/Florida/4/2006 and B/Malaysia/2506/2004) provided by the National Institute of Infectious Diseases, Japan
Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
Fetal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
Phosphate buffered saline without Ca²⁺ and Mg²⁺ (PBS)
Dulbecco’s Modified Eagle’s Medium F12 (DMEMF12) (Life Technologies, catalog number: 11320-033 )
Acetylated trypsin (Sigma-Aldrich, catalog number: T6763 )
Giemsa stain solution (Wako Pure Chemical Industries, catalog number: 079-04391 )
Methanol (Nacalai tesque, catalog number: 21915-93 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9576 )
Specific pathogen
Monoclonal antibody
MEM powder (Nisshin EM, catalog number: 05901 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
MES (Wako Pure Chemical Industries, catalog number: 344-08351 )
Fusion medium (see Recipes)

Equipment

96-well cell culture plate (Corning, catalog number: 3596 )
CO₂ incubator (37 °C, 5% CO₂)
Light microscope

Procedure

(Day 1) CV-1 cells in 100 μl MEM including 10% FBS are passaged to 96-well plate at 2.5 x 10⁵/ml and incubated in the CO₂ incubator for 24 h.
(Day 2) The cells are rinsed with 100 μl PBS once, adsorbed with 50 μl viruses in MEM at an MOI of 0.3 for 1 h in the CO₂ incubator.
The cells are rinsed with 100 μl PBS once, added 100 μl DMEMF12 including 0.2% BSA and incubated for 24 h in the CO₂ incubator.
(Day 3) The cells are rinsed with 100 μl MEM twice.
The cells in 100 μl MEM including 2.5 μg/ml acetyrated trypsin are incubated in the CO₂ incubator for 15 min.
They are rinsed with 100 μl MEM twice.
50 μl MEM containing different concentration of monoclonal antibody (concentration is 100, 25, 6.25, 1.5, 0.4 and 0.1 μg/ml) is incubated with the cells in different wells for 30 min in the CO₂ incubator.
After aspiration of medium, 100 μl fusion medium with each of pH is added and then incubated for 2 min in the CO₂ incubator.
The cells are rinsed with 100 μl MEM twice.
The cells are suspended with 100 μl DMEMF12 including 0.2% BSA and incubated in the CO₂ incubator for 3 h.
After aspiration of medium, the cells are fixed using 100 μl absolute methanol for 30 sec.
After aspiration of methanol, the cells are stained with 100 μl diluted Giemsa staining solution (1 drop per ml dH₂O) for 30 min at room temperature.
After rinsing the cells on tap water, the cells are observed by microscope (Figure 1).

Figure 1. Giemsa staining of CV-1 cells in fusion inhibition assay. Arrow heads show multinuclear giant cells.

Recipes

Fusion medium
Mix 2x MEM with 10 mM MES and 10 mM HEPES
Adjust to pH 5.0, 5.5, 6.0, 6.5 and 7.0
Mess up to 1x MEM
Autoclaved

Acknowledgments

This work was supported in part by the Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS) (http://www.jst.go.jp/global/kadai/h2011_thailand.html); and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science to MY (#23790660).

References

Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.