Abstract
Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). Surface localizations of IpaD and IpaB were monitored by FACS analysis according to the localization protocol for Pseudomonas aeruginosa homolog PcrV (Lee et al., 2010).
Keywords: Type 3 secretion system, Tip complex, Shigella, Flow cytometry
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Lee et al. (2010). This study was supported by grants from the Belgian Fonds National de la Recherche Scientifique Médicale (FRS-FNRS; Convention 3.4556.11) and from the European Community’s Seventh framework program FP7/2011–2015 under grant agreement No. 261742. L.S. and A.M are recipients of a PhD fellowship from the Belgian Fonds National de Recherches Industrielles et Agronomiques (FRIA). A part of this work was also supported by the Fonds Defay, and by Van Buuren and Héger-Masson foundations. We thank A. Op de Beeck for her help in performing the FACS experiment.
References
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Hi,you'll find the CR from VWR at this URL: https://uk.vwr.com/app/catalog/Product;jsessionid=6nvO4UhfRBxKo5Evg2WuAw**.node3?article_number=34140.184I haven't found the same product from US VWR...for CR agar plates' preparation :- prepare a 30 g/l TSB solution with bidistilled water and dissolve 15 g/l agar.- autoclave the medium for 15 min at 120°C (if you autoclave your medium for 20 min, sugar will caramelize and your plates will be darker, and even darkest after incubation with bacteria at 37°C)- Add the CR at a final concentration of 250 µg/ml (stock solution is prepared in bidistilled water at a concentration of 10 mg/ml) when the bottle can be safely handled with a protective gloveI hope it'll help you. Don't hesitate if you have more questions...Lionel