High-throughput measurement of dictyostelium discoideum macropinocytosis by flow cytometry

TY - JOUR

T1 - High-throughput measurement of dictyostelium discoideum macropinocytosis by flow cytometry

AU - Williams, Thomas

AU - Kay, Robert R.

N1 - Funding Information: We thank the Medical Research Council UK for core funding (U105115237) to RRK. Publisher Copyright: © 2018, Journal of Visualized Experiments. All rights reserved.

PY - 2018/9/10

Y1 - 2018/9/10

N2 - Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment. Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria. This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

AB - Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment. Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria. This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

KW - Biology

KW - Dictyostelium discoideum

KW - Endocytosis

KW - Flow cytometry

KW - Fluid uptake

KW - High-throughput

KW - Issue 139

KW - Macropinocytosis

KW - Single-cell resolution

UR - https://www.scopus.com/pages/publications/85054372368

U2 - 10.3791/58434

DO - 10.3791/58434

M3 - Article

C2 - 30247467

SN - 1940-087X

VL - 2018

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

IS - 139

M1 - e58434

ER -