Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

Summary

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

Abstract

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

Introduction

Macropinocytosis refers to the uptake of large quantities of extracellular fluid into membrane-bound cytoplasmic organelles called macropinosomes^1,2. It is a highly conserved process performed by free-living unicellular organisms, such as the amoeba Dictyostelium spp.³, as well as anthozoans⁴ and metazoans². In most cells, macropinocytosis is an induced event. The ligation of cell-surface receptors induces the protrusion of actin-driven plasma membrane extensions referred to as ruffles. A fraction of those ruffles, by some poorly ....

Protocol

1. Preparation of cells

1. Grow Raw264.7 cells at 37 °C and 5% CO₂ to 70% confluency in RPMI supplemented with 10% heat-inactivated serum.
2. One day before the assay, seed Raw264.7 cells at a density of 5 x 10⁴ cells per well in a 96-well plate. Ensure that each well contains 100 μL of growth medium. Ensure that the 96-well plate has black sides and a glass-bottom for imaging.
   NOTE: Here, 96-well plates were used for imaging. The 96-well plates allow for the use of smaller quantities of cells and reagents relative to larger imaging chambers. However, any chamber designed for imaging live cells can be used, and re....

Results

When measuring macropinosome pH, there is a period of time for which the dynamics of acidification cannot be measured. This period corresponds to the dextran loading phase (gray box in Figure 2C and Figure 3C,D) and will vary depending on the cell type used. The length of the loading phase will vary with (i) the macropinocytic activity of the cells and (ii) the sensitivity of the instrument used. It is recommended to adjust this period at the st.......

Discussion

Although there are a number of protocols for both low and high-throughput measurements of macropinocytic uptake in macrophages, fibroblasts, and even Dictyostelium spp.^3,7,31,32,33, very few attempts have been made to measure the luminal biochemistry of these dynamic compartments. This is likely due to a paucity of probes that can be efficiently targe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Black-walled 96 well plate	PerkinElmer	6005430
CypHer5e, NHS ester	Cytiva	PA15401
Dextran-amino 70 kDa	Invitrogen	D1862
DQ-ovalbumin	Invitrogen	D12053
FITC-dextran 70 kDa	Invitrogen	D1823
HBSS	Gibco	14287
Nigericin	Sigma Aldrich	N7143
OxyBurst Green-SE	Invitrogen	D2935
pHrodo Red, SE	Invitrogen	P36600
Raw264.7 cells	ATCC	TIB-71
RPMI medium	Gibco	11875093
SP5 Confocal Microscope	Leica	-
TRITC-dextran 70 kDa	Invitrogen	D1819
u-Dish	Ibidi	81156