Optimizing Lectin Staining Methodology to Assess Glycocalyx Composition of Legionella-Infected Cells
نویسندگان
چکیده
Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. utilizes effector proteins to hijack ER-Golgi vesicle trafficking sustain proliferation in its intracellular niche. has considerable influence on O-glycosylation but not N-glycosylation events the Golgi of infected cells. This research aims optimize use fluorescent lectins, which are bind carbohydrates, effectively label host-cell glycocalyx during infection. Methods: Epifluorescence imaging or flow cytometry were used lectin staining methodology. We noted most effective conditions for lectin-labeling when live HeLa cells incubated with lectins diluted Hank’s balanced salt solution (HBSS) 3% Bovine serum albumin (BSA) 10-30 minutes at 4 °C. Results: Incubating suspended necessitated smaller concentrations, whereas labeling adherent required considerably larger concentrations. Wheat germ agglutinin (WGA) mean fluorescence intensity (MFI) was concentration-dependent, Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did alter substantially increasing Discussion: The optimal concentration lectin-specific based whether assessed using epifluorescence. Furthermore, phosphate-buffered saline (PBS) dilution, cell permeabilization labelling, incubation fixed reduced productive labelling surfaces because it inhibited lectin's ability associated carbohydrate structure. Conclusion: Further diverse U937 macrophages necessary reach definitive conclusion effect overall composition infection these relevant immune
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ژورنال
عنوان ژورنال: Undergraduate Research in Natural and Clinical Science and Technology (URNCST) Journal
سال: 2023
ISSN: ['2561-5637']
DOI: https://doi.org/10.26685/urncst.490