By Christof R. Hauck (auth.), Steffen Rupp, Kai Sohn (eds.)
In contemporary many years, infectious ailments, as soon as believed to be relatively contained, became an important, resurgent box of study. In Host-Pathogen Interactions: equipment and Protocols, most sensible specialists learn the connection among the host and the pathogen, an important within the consequence of infection and the institution of disorder or asymptomatic, commensal colonization by means of organisms. The step by step laboratory equipment and protocols of this quantity learn host-pathogen interplay, with a spotlight on fungal, bacterial and parasitic pathogens, at a molecular point as a way to show the mechanisms of an infection and to spot the vulnerabilities of the pathogen of curiosity. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, the chapters characteristic short topic introductions, lists of the mandatory fabrics and reagents, and pointers on troubleshooting and fending off identified pitfalls.
Comprehensive and state-of-the-art, Host-Pathogen Interactions: equipment and Protocols serves as a simple access element for all these investigating the criteria liable for the pathogenicity of microorganisms.
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Extra info for Host-Pathogen Interactions: Methods and Protocols
Confluent cell layers with approximately 2 ϫ 105 cells are washed prior to infections with the infection medium, and the cells are infected with pneumococci in 500 L DMEM with HEPES (1x; PAA Laboratories) and 1% FBS per well Surface-Exposed Adherence Molecules of S. pneumoniae 37 with an MOI of 10 to 100 pneumococci per cell at 37°C under 5% CO2. 7. After the infection, the cells are rinsed several times with DMEM-HEPES or phosphate-buffered saline (PBS) to remove unbound bacteria. 5. Double Immunofluorescence Staining of Pneumococci and Microscopy 1.
6. Subsequently, unbound bacteria are eliminated by washing (1 to 3 times) the wells with PBS. 7. Fluorescence is measured at 485 nm/538 nm (excitation/ emission), and binding activity can be expressed as the percentage of the total applied pneumococci. When counting the number of applied FITC-labeled pneumococci after plating sample aliquots on blood agar plates, the number of pneumococci bound to the immobilized protein can also be calculated using the percentage of binding (18). 4. Cell Culture Infection Experiments with Streptococcus pneumoniae 1.
Ii. Add 5 L of the flow-through from each purified probe to 5 mL of scintillation fluid in separate scintillationcounter vials. iii. Count samples on the 33P channel. Multiply counts by a dilution factor of 20. Probes should yield a minimum of 5 ϫ 106 to 25 ϫ 106 cpm. 8. Discard flow-through fractions, columns, and elution tubes in the appropriate container for radioactive waste. 7. Hybridization Procedure 1. 1x SSC at 30°C for 5 min each time (see Note 15). 2. During the hybridization procedure, ensure that the printed surface of the microarray is facing up.