An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Overview

This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Purification of human polymorphonuclear leukocytes and isolation of human serum

NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit and should contain <25.0 pg/mL endotoxin to prevent unwanted priming of PMNs.

1. Bring 50 mL of 3% dextran-0.9% NaCl (w/v), 35 mL of 0.9% NaCl (w/v), 20 mL of 1.8% NaCl (w/v), 12 mL of 1.077 g/mL density gradient solution, and 20 mL of injection- or irrigation-grade water to room temperature.
2. To isolate human serum, incubate 4 mL of freshly drawn human blood without anti-coagulant at 37 °C in a 15 mL glass tube for 30 min. After incubation, centrifuge the sample at 2,000–3,000 × g for 10 min at room temperature. Transfer the upper serum layer into a fresh 15 mL conical centrifuge tube and place it on ice.
3. Combine 25 mL of freshly drawn heparinized (1000 units/mL) whole human blood with 25 mL of room temperature 3% dextran-0.9% NaCl (1:1 ratio) in two replicate 50 mL conical centrifuge tubes (50 mL total volume per tube). Mix by gently rocking each 50 mL conical tube and then let stand at room temperature for 30 min.
4. After incubation at room temperature, two separate layers will appear. Transfer the top layer of each dextran-blood mixture into new 50 mL conical tubes and centrifuge at 450 x g for 10 min at room temperature with low or no brakes.
5. Carefully aspirate both supernatants and discard them without disturbing the cell pellets. Gently resuspend each cell pellet in 2 mL of room temperature 0.9% NaCl, combine the resuspended pellets in a single 50 mL conical tube, then add the remaining 0.9% NaCl (final volume of 35 mL).
6. Carefully underlay 10 mL of room temperature of 1.077 g/mL density gradient solution beneath the cell suspension using a hand pipette. Spin at 450 x g for 30 min at room temperature with low or no brakes. Gently aspirate the supernatant without disturbing the cell pellet. The supernatant will contain peripheral blood mononuclear cells that can be collected as previously described.
7. Lyse the red blood cells by resuspending the cell pellet in 20 mL of room-temperature water. Mix gently by rocking the tube for 30 seconds. The lysis of red blood cells will be accompanied by a distinct decrease in turbidity.
8. Immediately add 20 mL of 1.8% NaCl (at room temperature [RT]) and centrifuge sample at 450 × g for 10 min at room temperature.
   NOTE: It is important to minimize the time that PMNs are left in water alone following red blood cell lysis to maximize PMN yield and prevent PMN lysis and/or activation.
9. Carefully aspirate the supernatant without disturbing the cell pellet. Gently resuspend the cell pellet in 2 mL of RT RPMI 1640 medium and place it on ice.
10. Count cells using a hemocytometer. Resuspend purified PMNs at a concentration of 1 x 10⁷ cells/mL with ice-cold RPMI and keep them on ice.
11. Combine 100 µL of purified PMNs (1 x 10⁶ cells) with 300 µL of ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 1 µL of propidium iodide stain in two replicate flow cytometry tubes. For a positive control for plasma membrane damage, add 40 µL of 0.5% Triton X-100 solution into one of the flow cytometry tubes and mix thoroughly.
12. Use flow cytometry to measure the forward scatter, side scatter, and propidium iodide staining (excitation/emission maxima at 535/617 nm) of purified cells (Figure 1).
    NOTE: Forward and side scatter analysis will identify unwanted populations of lymphocytes and monocytes. Propidium iodide will only stain cells with a compromised plasma membrane and purified PMNs that have pronounced populations of propidium iodide-positive cells should not be used. For these studies, purified PMNs were only used if they comprised >98% of purified cells and <5% stained positive for propidium iodide.
13. Prepare a 96-well plate for PMN cytotoxicity assays by coating individual wells that will be used in this assay with 100 µL of 20% isolated human serum that has been diluted with DPBS.
    NOTE: Plating PMNs directly on plastic or glass will cause activation of the cells. Be sure to include at least one negative control well that will only receive media and at least one positive control well that will receive 0.05% Triton X-100.
14. Incubate the plate at 37 °C for 30 min. Following incubation, wash the coated wells twice with ice-cold DPBS to remove any excess serum. Gently tap the plate upside down to remove any residual DPBS and place it on ice.
15. Gently add 100 µL of purified human PMNs at 1 x 10⁷ cells/mL to each coated well (1 x 10⁶ PMNs/well). Allow PMNs to settle in wells by incubating the plate on ice for at least 5 min. Keep the plate level to allow even distribution of cells in each well and leave it on ice to avoid unwanted activation of PMNs.

2. Cytotoxicity assay of S. aureus extracellular proteins against human polymorphonuclear leukocytes

1. Culture S. aureus overnight in tryptic soy broth (TSB) using a shaking incubator set at 37 °C. For these studies, 20 mL of TSB in separate 150 mL Erlenmeyer flasks were inoculated with frozen cultures of S. aureus strains USA300 or USA300ΔsaeR/S and grown for approximately 14 h with shaking at 250 rpm.
2. Subculture S. aureus by performing a 1:100 dilution of overnight bacterial culture with fresh media. Incubate at 37 °C with shaking until the bacteria reach the early stationary growth phase.
   NOTE: For these experiments, 20 mL of tryptic soy broth in 150 mL Erlenmeyer flasks were inoculated with 200 µL of overnight cultured USA300 or USA300ΔsaeR/S and incubated at 37 °C with shaking at 250 rpm for 5 h.
3. When bacteria have reached the early stationary growth phase, transfer 1 mL of subcultured S. aureus into a 1.5 mL microcentrifuge tube and centrifuge at 5,000 × g for 5 min at room temperature.
4. Following centrifugation, transfer the supernatant into a 3 mL syringe. Pass supernatants through a 0.22 µm filter and into a new 1.5 mL microcentrifuge tube on ice.
5. Perform serial dilutions of supernatants with ice-cold media used to culture S. aureus.
   NOTE: For the experiments shown, supernatants from USA300 and USA300ΔsaeR/S underwent four consecutive 1/2 log dilutions with ice-cold TSB.
6. Gently add supernatant samples or media alone (for negative and positive controls) to individual wells of 96-well plates containing PMNs on ice from step 1.15. For these experiments, 10 µL of USA300 or USA300ΔsaeR/S supernatant samples were added to each well. Gently rock plate to distribute supernatants in wells and incubate at 37 °C.
7. At desired times, remove the plate from the incubator and place it on ice. Add 40 µL of 0.5% Triton X-100 to the positive control well.
8. Gently pipette the samples up and down in each well to pull off all PMNs adhered to the plate completely, then transfer the samples to flow cytometry tubes on ice that contain 300 µL of ice-cold DPBS with 1 µL of propidium iodide.
9. Measure the proportion of propidium iodide-positive PMNs using flow cytometry (Figure 2A). When bound to DNA, propidium iodide has excitation/emission at 535/617 nm.

Representative Results

Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (<1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (shaded red).

Figure 2: Flow cytometry analysis of PMNs intoxicated with extracellular proteins produced by S. aureus. (A) Representative flow cytometry histogram of PMNs stained with propidium iodide after 30 min of incubation with media control (shaded blue), filtered USA300 supernatant at a final concentration of 1:110 (shaded grey), or 0.05% Triton X-100 (shaded red). (B) The proportion of propidium iodide positive PMNs after 30 min of incubation with different concentrations of USA300 or USA300∆saeR/S supernatants. (C) The proportion of propidium iodide positive PMNs over time following incubation with USA300 or USA300∆saeR/S supernatant at a final concentration of 1:110. Data are presented as mean ± SEM of at least 3 separate experiments with * p ≤ 0.05 and ** p ≤ 0.005 as determined by a two-tailed t-test.

Materials

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Injection, USP, 500 mL VIAFLEX Plastic Container	Baxter	2B1323Q	PMN purification
1.5 mL micro-centrifuge tubes with Snap Caps	VWR	89000-044	Used in washing cells
1.8% Sodium Chloride Solution	Sigma-Aldrich	S5150	PMN purification
12x75mm Culture tubes	VWR	60818-430	Used as flow cytometry tubes
20% (w/w) Dextran	Sigma-Aldrich	D8802	PMN purification
3125 Hand Tally Counter	Traceable Products	3125CC	For counting cells
50 mL conical centrifuge tubes	VWR	89039-656	For dispensing media
Bacto Tryptic Soy Broth, Soybean-Casein Digest Medium	FischerScientific	211823	For growing cell cultures
BD Disposable Syringes with Luer-Lok Tips, 3 mL	FischerScientific	BD 309657	For filtering supernatants
Bright-Line Hemocytometer	Sigma-Aldrich	Z359629	Cell counting apparatus
DPBS, 1x (Dulbecco's Phosphate Buffered Saline) with calcium and magnesium	Corning	21-030-CV	Used in washing cells
Ficoll-Paque PLUS	GE Healthcare	17-1440-02	PMN purification
Fisherbrand Sterile Polystyrene Disposable Serological Pipets	FischerScientific	13-678-11E	For aspirating liquid
Greiner CELLSTAR 96 well plates	Millipore Sigma	M0687	Plate for holding experimental samples
OMICRON Syringe Filters	Omicron Scientific	SFPV13R	For filtering supernatants
Propidium iodide	ThermoFisher Scientific	P3566	Membrane impermeable DNA stain
PYREX Brand 4980 Erlenmeyer Flasks	Cole-Parmer	EW-34503-24	For growing cell cultures
RPMI 1640, 1X without L-glutamine, phenol red	Corning	17-105-CV	Used in resuspending cells
Sterile Water for Irrigation, USP	Baxter	2F7113	PMN purification
The Pipette Pump	Bel-Art Products	F37898	For aspirating liquid
Triton X-100	Sigma-Aldrich	X100	Membrane integrity positive control