Le, Anh H;
Machesky, Laura M;
(2022)
Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells.
Bio-protocol
, 12
(7)
, Article e4367. 10.21769/bioprotoc.4367.
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Abstract
Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.
Type: | Article |
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Title: | Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.21769/bioprotoc.4367 |
Publisher version: | https://doi.org/10.21769/bioprotoc.4367 |
Language: | English |
Additional information: | This version is the version of record. For information on re-use, please refer to the publisher’s terms and conditions. |
Keywords: | Macropinocytosis, Macropinosomes, Dextran, CYRI-A, Fam49A, Cell migration, Actin |
UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology |
URI: | https://discovery.ucl.ac.uk/id/eprint/10187101 |
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