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EoE Sub Articles

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density at 600 nm (OD600) and concentration of bacteria must be determined empirically for the S. aureus strains to be tested before beginning this assay. The success of these experiments requires the consistent harvest of equal concentrations of each S. aureus</e...

cytotoxicity assay of staphylococcus aureus strains on neutrophils following phagocytosis

Source: Dankoff, J. G. et al., <a href="https://app.jove.com/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020)
In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

<img alt="Figure 1" src="/files/ftp_upload/21790/21790_Figure1.jpg" /> 
Figure 1: Flow cytometry analysis of PMNs following phagocytosis of S. aureus. (A) The proportion of propidium iodide positive PMNs 90 min after the phagocytosis of different concentrations of USA300 or USA300&#8710;saeR/S. (B) The concentration of opsonized S. aureus strains used for the experiments is shown in panel A. Data are presented as mean &#177;...

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density ...

Take virulent and less virulent Staphylococcus aureus strains.
Add serum to opsonize the bacteria.
Transfer increasing bacterial concentrations to chilled multi-well plate wells containing neutrophils. Low temperatures prevent neutrophil activation.
Centrifuge to facilitate synchronized phagocytosis.
Neutrophil receptors bind the opsonins, triggering bacterial internalization into phagosomes.
In virulent bacteria, the phagosomal environment triggers the expression of cytolytic toxins.
These toxins form pores, disrupting the phagosomes and compromising the neutrophil membrane integrity.
However, the less virulent bacteria exhibit reduced cytolytic toxin expression.
Post-incubation, detach the neutrophils and transfer them to flow cytometer tubes containing fluorescent propidium iodide (PI) dye.
The dye enters the neutrophils with compromised membranes and stains the DNA.
Using a flow cytometer, analyze the neutrophils for PI staining.
A concentration-dependent increase in the proportion of PI-positive neutrophils following phagocytosis of the virulent strain, as compared to the less virulent strain, indicates the cytotoxicity of the virulent strain against neutrophils.

cytotoxicity-assay-staphylococcus-aureus-strains-on-neutrophils

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

JoVE Journal

Immunology and Infection

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

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