Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells
| Autor: | Gianluca Scerra, Maria Gabriella Caporaso, Maurizio Renna, Massimo D'Agostino |
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| Přispěvatelé: | Scerra, Gianluca, Caporaso, MARIA GABRIELLA, Renna, Maurizio, D'Agostino, Massimo |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
chemistry.chemical_classification
Microscopy Microscopy Confocal Science (General) General Immunology and Microbiology Chemistry Histocytochemistry General Neuroscience Cell Culture Techniques Esters Cell Biology Size measurement General Biochemistry Genetics and Molecular Biology Amino acid Lysosomal compartment Q1-390 Biochemistry Molecular/Chemical Probes Protocol Humans Amino Acids Lysosomes Cells Cultured Fixation (histology) HeLa Cells |
| Zdroj: | STAR Protocols, Vol 2, Iss 4, Pp 100916-(2021) STAR Protocols |
| ISSN: | 2666-1667 |
| Popis: | Summary The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. For complete details on the use and execution of this protocol, please refer to Scerra et al. (2021). Graphical abstract Highlights • Methyl-group-modified amino acid analogs (LEU-ME) can localize to lysosomes • LEU-ME triggers a rapid expansion of the lysosomal compartment • Fixation procedure is a key step for preserving lysosomal morphology • Quantification and analysis of lysosomal dimensions The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment. We also present protocols to preserve lysosomal morphology, which yields a more accurate size measurement. |
| Databáze: | OpenAIRE |
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