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Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal <jats:italic>GBA<\/jats:italic> allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four <jats:italic>N<\/jats:italic>\u2010glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate\u2010polyacrylamide gel electrophoresis. Fluorescent activity\u2010based probes (ABPs) that covalently label GCase in reaction\u2010based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage\u2010like RAW264.7 cells. Specific attention was paid to the impact of 4\u2010(2\u2010hydroxyethyl)\u20101\u2010piperazineethanesulfonic acid\u00a0(HEPES) supplementation to bicarbonate\u2010buffered medium. Here, we report how HEPES\u2010buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES\u2010containing medium was also found to reduce maturation of other lysosomal enzymes (\u03b1\u2010glucosidase and \u03b2\u2010glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD\u2010patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.<\/jats:p>","DOI":"10.1002\/jcb.30234","type":"journal-article","created":{"date-parts":[[2022,3,21]],"date-time":"2022-03-21T11:58:58Z","timestamp":1647863938000},"page":"893-905","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["HEPES\u2010buffering of bicarbonate\u2010containing culture medium perturbs lysosomal glucocerebrosidase activity"],"prefix":"10.1002","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6458-1479","authenticated-orcid":false,"given":"Martijn J. 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