Highlights

  • Phagocytosis assaymeasuring the engulfment of bioparticles by RAW264.7 macrophages.
  • Quantification of phagocytosis by correlative fluorescence measurement and Quantitative Phase Imaging (QPI).
  • Automated analysis at the single-cell level eliminates confounding effects from cell proliferation and seeding density variations.

Introduction

Phagocytosis is the process by which a cell (e.g. macrophage, neutrophil or dendritic cell) engulfs
pathogenic or foreign bodies, giving rise to an internal compartment called the phagolysosome. Here, hydrolytic enzymes and the acidic environment break down the particles for disposal or recycling. It is one of the main mechanisms of the innate immune system and a primary response to infection.1

Macrophages exist in multiple states of readiness. In tissues, they are typically in a "resting" state where they slowly proliferate and clear up debris. Macrophage activation occurs when these resting macrophages receive chemical signals which alert them to the proximity of invaders. For example, Lipopolysaccharide (LPS), a component of the outer cell membrane of Gram-negative bacteria such as E. Coli, can be shed by these bacteria, and bind to receptors on the surface of macrophages. In response, these macrophages become activated and produce inflammatory cytokines, reactive oxygen and nitrogen species and begin phagocytosing the foreign bodies.

To understand macrophage activation, the regulation of phagocytosis, and the impact that the cellular environment has on phagocytosis, we must quantify particle engulfment. Traditionally, this was challenging but with the use of fluorescent bioparticles and recent developments in real-time
fluorescence microscopy it is possible to measure the total fluorescence to quantify macrophage
phagocytosis (Figure 1).2, 3 However, there are drawbacks with live fluorescencemicroscopy. For instance, phototoxicity is frequently encountered, which can impair sample physiology, and even lead to cell death.4 Furthermore, fluorescence microscopy typically measures total fluorescence intensity which can be misleading and inhibits the study of population heterogeneity.

Livecyte goes a step further by using label-free phase imaging with intermittent fluorescence to track individual cells and measure their fluorescence over time; this enables investigation of phagocytosis whilst substantially reducing phototoxicity effects. The high contrast label-free
images produced by Livecyte facilitate robust segmentation of cells and phagocytosis activity can be quantified by measuring individual cell fluorescence.

In this assay, we quantified phagocytosis using the RAW 264.7 macrophage-like cell line and pHrodo green E. coli bioparticles, which only fluoresce in the acidic environment of the phagosome (this method can easily be adapted to other phagocytes and phagocytic particles). In addition, as phagocytosis involves the actin-driven internalisation of particles, we sought to monitor the dose-response of the actin inhibitor cytochalasin D (10μM - 1nM) on phagocyte behaviour and morphology.

Hypothesis

  • Treating RAW cells with cytochalasin D, an actin inhibitor, will lead to a dose dependent reduced phagocytic activity.
  • Livecyte will be able to detect proliferative, and morphological changes to macrophages corresponding with phagocytic activity.
  • Using quantitative, correlative fluorescence detection Livecyte will be able to detect changes in rates of phagocytosis of pHrodo Green E. coli Bioparticles.

Method

Cell Culture

RAW 264.7 cells were routinely maintained in DMEM + 10% FBS + 1% NEAA (complete medium hereafter) at 37°Cwith 5%CO2/95% humidity prior to experiments. Cells were harvested using standard techniques and cell count and viability determined by trypan blue exclusion (ViCell; Beckman Coulter®). Cells were seeded into the wells of a 96-well plate at 1 x 104 cells/well and cultured overnight. Media was replaced with complete medium only or complete medium containing cytochalasin D (10μM - 1nM) and incubated for 1 hour prior to the addition of pHrodo Green E. coli BioParticles (10μg per well) and imaging. Positive (E. coli bioparticles, no cytochalasin D) and negative (no E. coli bioparticles, no cytochalasin D) controls were included.

Resources

  • RAW 264.7 cell line
  • DMEM (Gibco)
  • Non-Essential Amino Acid (NEAA)
  • Foetal Bovine Serum (FBS; Gibco)
  • Cytochalasin D (10μM - 1nM)
  • Invitrogen pHrodo Green E. coli BioPartciles
  • 96-well culture plate (Corning® 3603)
  • Livecyte Kinetic Cytometer (Phasefocus)
  • Livecyte Acquire & Analyse software (Phasefocus)

Time-lapse Imaging

High-contrast quantitative phase and fluorescence images were automatically captured using the
Livecyte Kinetic Cytometer for 24 hours at 13-minute intervals. Cells were imaged with an Olympus PLN 10X (0.25NA) objective and 1 mm x 1 mm field of view (FOV) per well. Three repeats were performed per treatment. Cells were maintained inside the Livecyte environmental chamber at 37°C with 5% CO2/95% humidity.

Analysis & Results

Quantitative Phase Images & Cell Segmentation

Livecyte automatically produces correlated quantitative phase and corresponding green fluorescence images. Figure 2 shows images at different time points during the 24-hour period and provides real-time visualisation of the internalisation of pHrodo green bioparticles when no inhibitor is present. Automated analysis of these images enables quantification of phagocytosis over time.

Cell Fluorescence

Livecyte’s Fluorescence Dashboard includes two line-plots: one showing the total fluorescence signal across the imaged region (Figure 3(A)) and the other showing the median fluorescence intensity within each cell over time (Figure 4).

An increase in fluorescence indicates internalisation of the bioparticles into the phagosome; therefore, it would be expected that the total fluorescence intensity should exhibit a cytochalasin D dose dependence; a decrease in total fluorescence intensity would be expected with increasing
concentrations of cytochalasin Ddue to inhibition of phagosome formation. However, this is not the case, as shown in Figure 3(A) where the total fluorescence is more than expected in the wells treated with 100nM of cytochalasin D.

The Proliferation Dashboard shows that the total cell count is significantly higher for these treatments (Figure 3(B)), implying more macrophages are present, resulting in more phagocytosis events and therefore a higher total integrated fluorescence intensity. Hence, a global measurement of total fluorescence intensity can be misleading if differing numbers of cells are present, making this read-out highly sensitive to initial seeding conditions and proliferation variations.

Figure 1: Phagocytosis and degradation of pHrodo green E. coli bioparticles.

Figure 2: Phase image of quantitative phase images with green fluorescence overlay. The images show the phagocytosis of pHrodo Green E. coli BioParticles using RAW 264.7 cells.

Figure 3: (A) Total integrated intensity of RAW 264.7 macrophages over time. No dose dependent response was observed. This is likely due to disparities in the initial cell count; (B): Cell count over duration of assay illustrates differences in starting cell count and accounts for lower relative total integrated intensities seen in 1nM and 10nM cytochalasin D in comparison to 100nM cytochalasin D. This shows how only observing population level data can lead to the masking of individual cell behaviour.

Figure 4: Livecyte can measure fluorescence expression at a single-cell level, to provide the median fluorescence intensity of the cells
over time. This is, therefore, independent of phagocyte cell count and here shows the predicted dose dependent phagocytic reduction upon increasing concentrations of cytochalasin D.

Download the Application Note pdf

Summary

Phasefocus's Livecyte generates high contrast quantitative phase images which are ideal for the automated segmentation and tracking of individual cells. Correlative fluorescence imaging can be acquired periodically, enabling the tracking of individual cell expression over long periods of time, in this
case providing quantification of phagocytosis activity. By using intermittent fluorescence, Livecyte substantially reduces phototoxicity effects whilst still being able to track individual phagocyte expression over time. Unlike a simple measurement of total fluorescence expression, this is independent of cell
number and can identify heterogeneity in a cell population.

In this short study, we have examined the effects of the actin inhibitor, cytochalasin D, on cell phagocytosis of pHrodo green E. coli bioparticles. Livecyte is able to observe individual cell behaviour, and therefore can measure fluorescence on a single-cell basis, thus removing assay sensitivity to initial
cell count and allowing individual macrophage phagocytosis to be measured independently from cellular proliferation.

Our Fluorescence Dashboard showed a dose-dependent reduction in the median fluorescence intensity of the cells, therefore reduction in cell phagocytosis, with cytochalasin D treatment. Livecyte's high content data generation enables time-lapse data for individual cells to be observed giving a far more indepth analysis of an experiment than end point, or total population metrics.


References

  1. Uribe-Querol & Rosales, 2017. Control of Phagocytosis by Microbial Pathogens. Front Immunol. 8. 1368
  2. Kapellos, Taylor, Lee, Cowley, James, Iqbal and Greaves, 2016. A novel real time Imaging platform to quantify macrophage phagocytosis. Biochem. Pharmacol. 116. 107-119
  3. Life Technologies, 2013. pHrodoTMRed and Green BioParticles®Conjugates for Phagocytosis. 1-6.
  4. Icha, Weber, Waters and Norden, 2017. Phototoxicity in live fluorescence microscopy, and how to avoid it. Bioessays. 39(8)