In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans
巨噬细胞对白色念珠菌的结合和促炎反应的体外分析方法
发布: 2014年05月05日第4卷第9期 DOI: 10.21769/BioProtoc.1123 浏览次数: 8535
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
Abstract
Macrophage recognition of Candida albicans (C. albicans) is facilitated by pattern recognition receptors that interact with the fungal pathogen associated molecular patterns (PAMPs). Dectin-1 is the major macrophage receptor that is known to recognize fungal Beta-glucans leading to induction of various immune responses. This receptor is also known to be required for in vivo protection against C. albicans (Taylor et al., 2007). We recently showed that the Dectin-1 mediated protection in vivo is strain-dependent, and that C. albicans can adapt to modulate immune recognition by Dectin-1 (Marakalala et al., 2013). In vitro analysis, however, showed a Dectin-1-dependent and pro-inflammatory responses against all strains tested. This protocol describes in detail the in vitro analysis used in the paper. In particular, methods involved in fluorescent labeling of live C. albicans, quantification of macrophage binding of the pathogen, and pro-inflammatory responses to yeast and hyphal forms of the fungi are described.
Keywords: Candida albicans (白色念珠菌)
Dectin-1 (树突状细胞相关性C型植物血凝素1)
Beta-glucans (β-葡聚)
Macrophages (巨噬细胞)
Pro-inflammatory responses (炎性反应)
Materials and Reagents
- Candida albicans strains, SC5314 and ATCC18804
- Mice
Note: 2x per WT or Dectin-1 KO mice on C57BL/6 background should give sufficient macrophages for a full 24-well plate experiment. Mice were age and sex matched.
- Thioglycollate-elicited macrophages
Note: This method has been described in detail in Kerrigan et al. (2012).
- RPMI 1640 medium (Life Technologies, catalog number: 11875-093 )
- 70% ethanol
- Sabouraud Dextrose broth (Oxoid Limited, catalog number: CM0147 )
- Rhodamine Green-X (Life Technologies, InvitrogenTM, catalog number: R-6113 )
- Difco Thioglycollate Broth (BD Biosciences, catalog number: 225710 )
- Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
- OptEIA TNF kit (BD Biosciences, catalog number: 555268 )
- Zymosan (Life Technologies, InvitrogenTM, catalog number: Z2849 )
- Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
- Fetal Bovine/Calf Serum (FCS) (Sigma-Aldrich, catalog number: F6178 )
- EDTA (Sigma-Aldrich, catalog number: E9884 )
Equipment
- Titer-Tek Fluoroskan II (Labsystems)
- Shaker
Note: The shaker temperature was controlled at 30 °C and the speed was between 150 to 200 rpm.
- Centrifuge (Eppendorf, model: 5810R with swing bucket rotor S-4-72 )
- Light microscope
- Haemocytometer
- 24-well plates (BD Biosciences)
- 2 ml Eppendorf tube
- 96-well plates (black in color for fluorescence measurement) (BD Biosciences)
- Rubber back of syringe plunger
Note: The make or brands for above equipment are not important for reproducibility.
Procedure
文章信息
版权信息
© 2014 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:
- Marakalala, M. J. (2014). In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans. Bio-protocol 4(9): e1123. DOI: 10.21769/BioProtoc.1123.
- Marakalala, M. J., Vautier, S., Potrykus, J., Walker, L. A., Shepardson, K. M., Hopke, A., Mora-Montes, H. M., Kerrigan, A., Netea, M. G., Murray, G. I., Maccallum, D. M., Wheeler, R., Munro, C. A., Gow, N. A., Cramer, R. A., Brown, A. J. and Brown, G. D. (2013). Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1. PLoS Pathog 9(4): e1003315.
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分类
微生物学 > 微生物-宿主相互作用 > 真菌
免疫学 > 宿主防御 > 鼠
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