Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Summary

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Abstract

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca²⁺. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.

To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Introduction

Neutrophils (PMNs) are professional phagocytes that play a key role in the innate immune response against Staphylococcus aureus ¹. The constant battle between host and microbe has led to an arms race of both. Recently, community-associated (CA) strains of meticillin resistant S. aureus (MRSA) have emerged that seem to be very efficient in circumvention of neutrophil killing ^2,3. Excessive phenol soluble modulin (PSMs) production of CA-MRSA has been associated with higher virulence ^4,5. Human neutrophils can recognize these PSMs via FPR2 which lead to activation of this G-protein coupled receptor ⁶. One of t....

Protocol

1. Isolation of PMNs From Human Blood by Density Centrifugation

1. Draw 5 9 ml tubes of heparinized venous blood.
2. Prepare dual layer Ficoll gradients (4 gradients for 5 tubes blood) as follows: Pour 12 ml of a density 1.119 g/ml ficoll solution in a 50 ml tube and carefully layer 10 ml of a density 1.077 g/ml ficoll solution on top.
3. Dilute the blood with an equal volume of PBS.
4. Layer the diluted blood carefully on the dual layer Ficoll gradient; 20-25 ml per gradient.
5. Centrifuge 20 min at 396 x g in a swinging bucket rotor, 22 °C without braking.
6. Prepare cold RPMI containing 0.05% Human Serum Albumin (RPMI....

Results

Flow cytometric assay for assessment of calcium mobilization in human PMNs

Incubating neutrophils with a concentration series of synthetic PSMα3 resulting in rapid activation as measured by calcium flux, which is shown by an increase in the signal in FL-1. Pre-incubation of synthetic PSMα3 with 0.01%, 0.1% or 1% human serum significantly inhibited the ability to elicit calcium fluxes (Figure 1).

Analysis of GFP expression in bacteria after phagocytosis by.......

Discussion

In the methods described here some steps are very critical. We will highlight these here.

For the isolation of neutrophils by density gradient centrifugation it is important not to disturb the layers during or after the centrifugation steps. When aspirating the neutrophils using a plastic pipette, make sure not to squeeze the balloon while in the layer of cells, as ejecting liquid will disturb the layers. Also, visually inspect the pellet of neutrophils after the osmotic shock and centrifugat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluo-3, AM	Molecular Probes / Life Technologies	F-1241
Ficoll-Paque	GE Healthcare	17-5442-03	density 1.077 g/ml
Histopaque	Sigma	11191	density 1.119 g/ml
RPMI 1640	Gibco, Life Technologies	52400-025	contains 25 mM HEPES and L-glutamine
Leica TCS SP5 microscope	Leica Microsystems, The Netherlands	TCS SP5	objective: HCX PL APO 40x/0.85
FACSCalibur	BD Biosciences	FACSCalibur	Very important that the tube can be removed and replaced during the measurement process