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Measurement of IFN-α Subtype Concentrations (Virus-free, Cell-based Bioassay)

IFN-α亚型浓度的测定（无病毒，基于细胞的生物分析）

Kathrin Gibbert Kathrin Gibbert

发布: 2013年06月20日第3卷第12期 DOI: 10.21769/BioProtoc.803 浏览次数: 8977

评审: Fanglian HeLin Fang

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Aug 2012

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Abstract

The induction of type I IFN is the immediate host response against viral infections. Type I IFNs belong to a multigene family including up to 14 different IFN-α subtypes and one IFN-β. They are highly conserved and bind the same receptor (IFNAR1/2) with varying affinities, although they differ in their biological activities.

Keywords: Type I IFNs (I型IFNs) search IFN-alpha subtypes (IFN-α亚) search Mx expression (MX的表达) search

Materials and Reagents

  1. 7AAD (7-amino-actinomycin D) (BD Pharmingen, catalog number: 51-68981E )
  2. Bovine serum albumin (BSA) (PAA Laboratories GmbH, catalog number: K41-001 )
  3. DMEM (Life Technologies, Gibco®, catalog number: 41966-029 )
  4. Superior FBS (fetal bovine serum, not heat-inactivated) (Biochrom, catalog number: S0615 )
  5. Mx/RAGE7 cells (virus-transformed adherent cell line with a temperature-inducible promotor; must be cultured at 32 °C; cells express the Mx transgene and a promotorless eGFP gene which is expressed due to type I IFN stimulation ) (Bollati-Fogolin and Muller, 2005)
  6. PBS (Life Technologies, Gibco®, catalog number: 14190-136 )
  7. Penicillin/streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
  8. Propidium iodide (eBioscience, catalog number: 00-6990-50 )
  9. Murine IFN-α (PBL, catalog number: 12100-1 )
  10. Sodium azide (Applichem, catalog number: A1430.0010 )
  11. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360-039 )
  12. Trypsin EDTA (PAA Laboratories GmbH, catalog number: L11-004 )
  13. β-mercaptoethanol (Life Technologies, Gibco®, catalog number: 31350-010 )
  14. Media for Mx/RAGE7 cells (see Recipes)
  15. FACS buffer (see Recipes)

Equipment

  1. 96-well flat bottom plate (Falcon BD Labware, catalog number: 3072 )
  2. 1.5 ml microfuge tubes
  3. FACS tubes (BD Biosciences, Falcon®, catalog number: 352054 )
  4. Flow cytometer (e.g. BD LSR II)
  5. Incubator (37 °C; 5% CO2)
  6. Incubator (32 °C; 5% CO2)

Procedure

English
中文翻译

文章信息

版权信息

© 2013 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:

  1. Gibbert, K. (2013). Measurement of IFN-α Subtype Concentrations (Virus-free, Cell-based Bioassay). Bio-protocol 3(12): e803. DOI: 10.21769/BioProtoc.803.
  2. Gibbert, K., Joedicke, J. J., Meryk, A., Trilling, M., Francois, S., Duppach, J., Kraft, A., Lang, K. S. and Dittmer, U. (2012). Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection. PLoS Pathog 8(8): e1002868.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物-宿主相互作用 > 病毒

免疫学 > 免疫细胞功能 > 细胞因子

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