Viral Immunofluorescence with Rift Valley Fever Virus Infected MEFs in a 96 Well Plate
在96孔板中用病毒免疫荧光法检测受裂谷热病毒感染的小鼠胚胎成纤维细胞（MEF）
发布: 2012年12月05日第2卷第23期 DOI: 10.21769/BioProtoc.297 浏览次数: 11057
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参见作者原研究论文
The authors used this protocol in:
Apr 2012
Abstract
Immunofluorescence is a method to detect viral infection in multiple types of host cells. This procedure can be adapted for both high-throughput and low-throughput assays for any virus for which there are antibodies available. Time of infection and virus multiplicity of infection (MOI) vary and should be optimized for each virus and host cell type. Here we give an example of viral immunofluorescence in a 96 well plate with a Rift Valley fever virus (RVFV, strain MP12) infection in mouse embryonic fibroblasts (MEF).
Materials and Reagents
- Mouse embryonic fibroblasts (or other host cell)
- Rift Valley fever virus MP12 (or other virus)
- Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965-084 )
- Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
- Penicillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
- L-Glutamine (Life Technologies, Gibco®, catalog number: 35050-061 )
- Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies, Gibco®, catalog number: 14190-136 )
- 0.05% Trypsin (Life Technologies, Gibco®, catalog number: 25300-054 )
- Ethanol (Sigma-Aldrich, catalog number: 459844 )
- Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5368 )
- Formaldehyde solution (Sigma-Aldrich, catalog number: 252549 )
- TritonX-100 (Sigma-Aldrich, catalog number: 78787 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2158 )
- Primary antibody: Mouse anti-RVFV ID8 (obtained privately from C. Schmaljohn USAMRIID)
- Secondary antibody: AlexaFluor goat anti-mouse 488 (Life Technologies, Invitrogen™, catalog number: A11029 )
- Hoechst 33342 (Sigma-Aldrich, catalog number: B2261 )
- Plate sealing film (Denville Scientific, catalog number: B1212-4 )
- Complete DMEM (see Recipes)
- Fixative (see Recipes)
- PBST (see Recipes)
- Block (see Recipes)
Equipment
- Black, clear-bottomed 96 well plates (Corning Incorporated, catalog number: 3712 )
- T75 flask for cell maintenance (Corning Incorporated, catalog number: 430725 )
- Type II Laminar Flow Hood (ESCO Corporation)
- Thermo Forma Series II 37 °C Incubator with 5% CO2 (Thermo Fisher Scientific, Forma, catalog number: 3110 )
- Hemocytometer (Hausser Scientific Brightline)
- Multichannel pipette (Fisherbrand, catalog number: FJ19506 )
- Matrix Well Mate (optional) (Thermo Fisher Scientific)
- Plate shaker (Barnstead International, catalog number: 4625 )
- Centrifuge (Eppendorf, catalog number: 5810R )
- Multichannel Manifold Aspirator (Drummond, catalog number: 3-000-096 )
- Sterile basins (Thermo Fisher Scientific, catalog number: 13681500 )
- Quatracide (Thermo Fisher Scientific, catalog number: 50200423 )
- Automated inverted microscope (Molecular Devices Image Express Micro)
Software
- Image Analysis software (MetaXpress: Molecular Devices Version 2.0.0.13)
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Moser, T. S. and Cherry, S. (2012). Viral Immunofluorescence with Rift Valley Fever Virus Infected MEFs in a 96 Well Plate. Bio-protocol 2(23): e297. DOI: 10.21769/BioProtoc.297.
- Moser, T. S., Schieffer, D. and Cherry, S. (2012). AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog 8(4): e1002661.
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分类
免疫学 > 免疫细胞染色 > 免疫检测
微生物学 > 微生物-宿主相互作用 > 病毒
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