A total of 1.25 × 105 Huh-7 cells were first seeded on coverslips in 24-well plates (TPP Techno Plastic Products) for 24 h. Plasmids encoded with the spike of EMC/2012, BF785, or an empty vector were transfected in the cells using TransIT-LT1 transfection reagent (Mirus) (25 (link)). At 20 h posttransfection, the cells were fixed with 4% PFA, permeabilized with 0.3% Triton X-100, and immunolabeled using primary polyclonal antibody against MERS-CoV spike protein (SinoBiological) and secondary Alexa Fluor 488-conjugated goat anti-rabbit antibody (Thermo Fisher). Nuclei were stained using DAPI. Images were acquired by a Zeiss LSM710 inverted confocal microscope with a 20× objective. Cell scoring function in Metamorph software was used to quantify syncytia formation by calculating nuclei/syncytium.
Protocol detail
Visualizing MERS-CoV Spike Protein Expression
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Alexa fluor 488
Anti antibody
Antibody
Cell function
Cells
Confocal microscope
Dapi
Goat
Mers cov
Nuclei
Plasmids
Rabbit
Spike protein
Syncytia
Transfection
Triton x 100
Vector
Corresponding Organization :
Other organizations : University of Hong Kong, HKU-Pasteur Research Pole, National Veterinary Institute, Kenya Wildlife Service, African Union, Food and Agriculture Organization of the United Nations, Animal, Santé, Territoires, Risques et Ecosystèmes, Département Santé Animale, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Institut Pasteur du Cambodge, Maladies Infectieuses et Vecteurs: Écologie, Génétique, Évolution et Contrôle, Centre National de la Recherche Scientifique, Centre National de la Recherche Scientifique et Technologique, Institut Agronomique et Vétérinaire Hassan II, Guangzhou Medical University, First Affiliated Hospital of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Hong Kong Jockey Club, Chinese University of Hong Kong
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Variable analysis
independent variables
- Plasmids encoded with the spike of EMC/2012
- Plasmids encoded with the spike of BF785
- Empty vector
- Syncytia formation quantified by calculating nuclei/syncytium
- Total of 1.25 × 10^5 Huh-7 cells seeded
- Cells seeded on coverslips in 24-well plates (TPP Techno Plastic Products)
- Cells incubated for 24 h before transfection
- Transfection reagent used: TransIT-LT1 (Mirus)
- Cell fixation with 4% PFA at 20 h posttransfection
- Cell permeabilization with 0.3% Triton X-100
- Primary antibody: polyclonal antibody against MERS-CoV spike protein (SinoBiological)
- Secondary antibody: Alexa Fluor 488-conjugated goat anti-rabbit antibody (Thermo Fisher)
- Nuclei stained using DAPI
- Imaging by Zeiss LSM710 inverted confocal microscope with 20× objective
- Positive control: Not explicitly mentioned
- Negative control: Empty vector
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