Protocol Considerations for In Vitro Metabolic Glycoengineering of Non-Natural Glycans.

Metabolic glycoengineering (MGE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides, where they can modulate a host of biological activities or be exploited as tags for bioorthogonal and chemoselective ligation reactions. Over the past decade, azido-modified monosaccharides have become the go-to analogs for MGE; at the same time, analogs with novel chemical functionalities continue to be developed. Therefore, one emphasis of this article is to describe a general approach for analog selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell-surface glycans have been successfully remodeled by MGE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. This manuscript concludes by detailing how one of these detection methods-flow cytometry-can be successfully utilized to quantify MGE analog incorporation and set the stage for numerous follow-up applications. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Incubation of cells with sugar analogs Support Protocol: Routine growth and maintenance of Jurkat cells Basic Protocol 2: Cell viability assays Basic Protocol 3: Periodate-resorcinol assay to measure analog uptake and incorporation into metabolic pathways Basic Protocol 4: Quantitation of cell-surface glycoconjugates.