High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Summary

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Abstract

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Introduction

Quantification of fluorescent signal has been widely used in a multitude of scientific methods ranging from PCR, flow cytometry, confocal microscopy, and FRET analysis to multiplex ELISA. Fluorescence imaging and quantification has a broad application and can be a great tool for quantitative analysis of various cellular processes. Use of fluorescent markers and their signal has been revolutionized in the last decade, and emergence of fluorescent plate readers has facilitated the high throughput quantification of fluorescence emitted during cellular processes.

Fluorometric analysis can serve as a great tool in quantification of phagocytosis.....

Protocol

NOTE: This protocol can be used for multiple applications such as quantification of phagocytosis and actin polymerization as used in our previously published work¹². The protocol lends itself to a variety of modification, and Table 1 lists cell types and particles successfully utilized in past studies. Standard use of this protocol for quantification of phagocytosis or actin polymerization is illustrated in Diagram 1.

Results

Two major ways of quantifying phagocytosis and the subsequent actin polymerization through the use of this protocol is to observe continuous or synchronized phagocytosis.

Microscopic analysis (Figure 1B) illustrates fluorescent images comparable to what the fluorometer is recording (also see Diagram 1). In Figure 1B are red fluorescence of actin, green 268 fluorescence of FITC labels HKOP E. coli, nuclear DAPI stain, and the merged im.......

Discussion

Major limitations of the fluorometric technique are experimental variance as well as cell loss associated with extensive washing and use of non-adherent cells.

Variance is observed largely due to the variation in fluorescent particles as weighing and pipetting during preparation of the stock solution is not an exact way of maintaining identical numbers of particles from experiment to experiment. To address the problem of variance between experiments, data can be expressed in relative terms, fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 μm yellow-green fluorescent Fluo-Spheres;	Molecular Probes	F8852	combine 50μl with 50μl opsonizing reagent for 30 min at 37oC before use
Heat killed E. coli BioParticles fluorescein conjugate	Molecular Probes	E2861	reconstitute (5 mg) in 50 μl of PBS and combine with 50 μl opsonizing reagent for 30 min at 37 °C before use
Opsonizing reagent	Molecular Probes	E2870
Rhodamine phalloidin	Molecular Probes	R415
DAPI	Sigma-Aldrich	D9542
Trypan blue	Gibco	15250-061
The items below are available in many brands but the items we used in this study are from the following manufacturers:
RPMI Cell growth media	Gibco		Supplemented with 10% FBS and 1% pennicillin/streptomycin; warm in 37 °C before use
Fluorescent plate reader - Fluostar Omega	BMG Labtech
Paraformaldehyde (16%)	Fischer Scientific	AA433689M	dilute to 4% before use
96-well plates	Greiner	655097	clear or black or clear bottom - black plates
Multichannel pipette (8 - 12 channels)
Reagent reservoirs
1x PBS
Microfuge tubes (0.6 ml)
Conicles (10 ml)