High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry.

Thomas Williams,Robert R Kay

doi:10.3791/58434

Abstract

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.

Highlights

Large-scale non-specific fluid uptake by macropinocytosis is important in several biological contexts[1], including antigen sampling by immune cells[2], pathogen entry into host cells[3], cancer cell proliferation[4] and the spreading of prion diseases[5]
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
Whereas other methods to assess fluid uptake are low throughput, washing the cells in situ and the use of sodium azide to detach cells are the critical steps in this method, which allow high-throughput measurement of macropinocytosis, membrane uptake, or phagocytosis by Dictyostelium

Summary

Introduction

Large-scale non-specific fluid uptake by macropinocytosis is important in several biological contexts[1], including antigen sampling by immune cells[2], pathogen entry into host cells[3], cancer cell proliferation[4] and the spreading of prion diseases[5]. The standard technique used to measure macropinocytosis by mammalian cells involves fixing cells after pulsing with dextran for a short period of time followed by microscopy to determine the area of a cell that is occupied by dextran-positive vesicles[18] This technique does not account for the possibility of macropinosomes shrinking upon entering the cell, which has been reported in Dictyostelium[19], and only takes into account single planes of the cell, meaning the volume internalized is unclear. Cells are shaken at high density in fluorescent dextran and samples removed at various time-points for determination of the internalized fluorescence using a fluorimeter Cells prepared this way can be analyzed by flow cytometry to gain single cell, rather than population-level, resolution[22], this remains low-throughput. This method was designed to overcome the limitations of the above methods and allow simultaneous comparison of the fluid uptake of large numbers of strains/conditions while using fewer resources and reducing the labor involved

Preparation of Cells and Materials

Performing Fluid Uptake Time-courses

Dose Response Curves

Phagocytosis and Membrane Uptake

Representative Results

Discussion