%I Bio-Protocol, LLC %J Bio-protocol %L discovery10187101 %K Macropinocytosis, Macropinosomes, Dextran, CYRI-A, Fam49A, Cell migration, Actin %T Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells %N 7 %D 2022 %V 12 %O This version is the version of record. For information on re-use, please refer to the publisher’s terms and conditions. %A Anh H Le %A Laura M Machesky %X 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.