Staphylococcus aureus is a gram-positive bacteria that produces a wide range of virulence factors that promote pathogenesis. These include bi-component leukotoxins that disrupt the membrane integrity of polymorphonuclear leukocytes, also called PMNs or neutrophils. This video illustrates the isolation of PMNs from human whole blood and two distinct assays for quantifying S.aureus cytotoxicity against these important innate immune cells.
These assays can all be accomplished with standard laboratory equipment and are easily adaptable to examine other aspects of host pathogen interaction. These experiments will use whole blood drawn from human donors and will require approval from the appropriate institutional committee or review board as well as a statement that informed consent was obtained from all participants. The first step of this procedure is the isolation of neutrophils through the use of centrifugation and density gradients.
First, bring 50 milliliters of 3%dextran 0.9%sodium chloride, 35 milliliters of 0.9%sodium chloride, 20 milliliters of 1.8%sodium chloride, 12 milliliters of 1.077 grams per milliliter ficoll solution and 20 milliliters of water to room temperature. Transfer 25 milliliters of freshly drawn heparinized whole human blood into two conical centrifuge tubes. Combine 25 milliliters of freshly drawn heparinized whole blood with 25 milliliters equivalent volume of room temperature, 3%dextran, 0.9%sodium chloride in a one-to-one ratio.
Mix by gently rocking each 50 milliliter conical tube and then let stand at room temperature for 30 minutes. After incubation at room temperature, two separate layers will appear. Transfer the top layer of each dextran blood mixture into new 50 milliliter conical tubes.
Centrifuge at 450 g for 10 minutes at room temperature with low or no breaks. Carefully aspirate both supernatants and discard without disturbing the cell pellets. Gently resuspend each cell pellet in two milliliters of 0.9%sodium chloride.
Combine the resuspended pellets in a single, 50 milliliter conical then add the remaining 0.9%sodium chloride to a final volume of 35 milliliters. Carefully underlay 10 milliliters of 1.077 grams per milliliter ficoll solution beneath the cell suspension using a hand pipette. After spinning at 450 g for 30 minutes at room temperature, with low or no breaks, gently aspirate the supernatant without disturbing the cell pellet, as previously shown.
Lyse the red blood cells by resuspending the cell pellet in 20 milliliters of room temperature water. Mix gently by rocking for 30 seconds. Immediately add 20 milliliters of 1.8%sodium chloride and centrifuge sample at 450 g for 10 minutes at room temperature.
Carefully aspirate supernatant as shown before. Gently resuspend the cell pellet in two milliliter room temperature RPMI and place on ice. Count cells using a hemacytometer.
Resuspend purified PMNs at a concentration of one times 10 to the 7th cells per milliliter with ice cold RPMI and keep on ice. Combine 100 microliters of purified PMNs with 300 microliters of DPBS, containing one microliter of propidium iodide stain in two replicate flow cytometry tubes. For a positive control, add 40 microliters of 0.5%Triton X-100 solution into one of the flow cytometry tubes and mix thoroughly.
Use flow cytometry to measure the forward scatter, side scatter, and propidium iodide staining to determine the purity and integrity of isolated PMNs. Only PMN preparations that are above 98%pure and above 95%propidium iodide negative should be used. For the protocol outlining the isolation of normal human serum, refer to the full manuscript.
Prepare a 96-well plate for PMN cytotoxicity assays by adding 100 microliters of 20%isolated human serum diluted with DPBS to individual wells that will be used in this assay. 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.Incubate the plate at 37 degrees Celsius for 30 minutes. Following the incubation, wash the coded wells two times with 100 microliters of ice cold DPBS and place plate on ice.
Remove any residual DPBS from plate wells and gently add 100 microliters of purified human PMNs at one times 10 to the 7th cells per milliliter to each coded well. Allow plated neutrophils to settle by leaving them on ice for at least five minutes before use in the following cytotoxicity assays. This section describes an assay that quantifies the cytotoxicity of extracellular proteins produced by staph aureus against human PMNs.
Culture S.aureus overnight in appropriate media using a shaking incubator set at 37 degrees Celsius the day prior to the experiment. Subculture S.aureus by performing a one to 100 dilution of overnight bacteria cultures with fresh media. Incubate at 37 degrees Celsius with shaking until bacteria reach early stationary growth phase.
After five hours of growth, transfer one milliliter of subculture S.aureus into a 1.5 milliliter microcentrifuge tube and centrifuge at 5, 000 g for five minutes at room temperature. Following centrifugation, aspirate supernatants. Pass aspirated supernatants through a 0.22 micron syringe filter into a new 1.5 milliliter microcentrifuge tube and place on ice.
Perform serial dilutions of supernatants with ice cold media used to culture Staph aureus. Gently add supernatant samples or media loaned for negative and positive controls to individual wells of a 96-well plate containing PMNs on ice, as described earlier. Gently rock plate to distribute supernatants in wells and incubate at 37 degrees Celsius.
At desired times, remove plate from incubator and place on ice. Add 300 microliters of ice cold DPBS containing one microliter of propidium iodide to each flow cytometry tube. Transfer the samples to flow cytometry tubes on ice.
For the positive control well, add 20 microliters of 10%Triton X to the sample prior to transferring to a flow cytometry tube. Analyze propidium iodide staining of intoxicated PMNs using flow cytometry. This section describes an assay that quantifies the cytotoxicity of S.aureus following phagocytosis by human PMNs.
Growth curves defined by the optical density at 600 nanometers and the concentration of bacteria must be determined for the relevant Staph aureus strains prior to use in this assay. Start overnight cultures of S.aureus strains and subculture bacteria, as described previously. Once subcultured Staph aureus has reached mid-exponential growth, transfer one milliliter of culture bacteria to 1.5 milliliter microcentrifuge tube and centrifuge at 5, 000 g for five minutes at room temperature.
Wash S.aureus by aspirating its supernatant without disturbing the pellet and resuspend the pelleted bacteria at one milliliter DPBS. Vortex the samples for 30 seconds. Centrifuge the samples at 5, 000 g for five minutes at room temperature.
To oxenize Staph aureus, first resuspend the bacterial pellet in one milliliter of 20%human serum. Then incubate samples at 37 degrees Celsius with agitation for 15 minutes. Wash oxenized Staph aureus following centrifugation as shown previously.
Dilute oxenized Staph aureus strains to one times 10 to the 8th colony-forming units, CFUs per milliliter, with ice cold RPMI. Vortex the sample for 30 seconds and place on ice. Confirm the concentrations of Staph aureus used for these assays by plating one to 10 serial dilutions of bacteria on agar.
Gently add 100 microliters per well of each Staph aureus strain, or RPMI for positive and negative controls, to PMNs in a 96-well plate on ice from step 1.14. Gently rock plate to distribute Staph aureus in wells. Synchronize phagocytosis by centrifuging plate at 500 g for eight minutes at four degrees Celsius and incubate plate at 37 degrees Celsius immediately following centrifugation as time zero.
At the desired time points, put the plate on ice and add 200 microliters of ice cold DPBS containing one microliter propidium iodide to each flow cytometry tube then transfer samples to their corresponding tube. Analyze samples for propidium iodide staining using flow cytometry as described in 2.8 and 2.9. The transcription of bi-component leukotoxins that target human PMNs by Staph aureus requires the SA or S2 component system to demonstrate the utility of the described assays for quantifying Staph aureus cytotoxicity against human PMNs.
These experiments were performed with a clinically relevant Staph aureus isolate identified as USA300 in an isologenic deletion mutant of SA or S in this strain. The left panel depicts purified PMNs and the right panel depicts intentionally contaminated samples as an example. PMNs isolated using the procedures described in section one of this protocol were stained with propidium iodide and examined using flow cytometry.
Forward and side scatter plots were used to detect contamination of purified PMNs by monocytes or lymphocytes for years 1A and 1B. PMN integrity was determined using propidium iodide staining. By using the described method of human PMN purification, we can consistently isolate five times 10 to the 7th to one times 10 to the 8th PMNs that are over 98%pure and are over 95%propidium iodide negative.
The cytotoxicity of extracellular proteins produced by USA300 and the AsaeR/S mutant were tested against purified PMNs. These experiments demonstrate a concentration dependent increase in the propidium iodide staining of purified PMNs following 30 minutes of intoxication with extracellular proteins produced by USA300, but not with the AsaeR/S mutant. Further experiments demonstrated a steady increase in the proportion of propidium iodide positive PMNs following intoxication by USA300 extracellular proteins that plateaued after approximately 30 minutes.
Minimal propidium iodide staining of human PMNs was noted at all time points following exposure to extracellular proteins produced by the AsaeR/S mutant. Last, the cytotoxicity of USA300 and AsaeR/S mutant against PMNs following synchronized phagocytosis was tested, a concentration dependent increase in the proportion of propidium iodide positive PMNs was observed 90 minutes after the phagocytosis of USA300. Significantly fewer PMNs were propidium iodide positive following the phagocytosis of the AsaeR/S mutant.
Enumeration of the USA300 and AsaeR/S mutant inoculum was used in each of these experiments demonstrated that the contrast in cytotoxicity between these strains was not due to differences in the concentration of bacteria used. This protocol describes the purifications of PMNs from human blood in two distinct techniques for quantifying the cytotoxicity of S.aureus against these innate immune cells. The success of these procedures will depend on the quality of the purified PMNs and the appropriate preparation of Staph aureus bacteria or supernatants.
Please consult the full manuscript for important points to consider while performing these procedures. USA300 is a virulent MRSA isolate and the loss of AsaeR/S dramatically reduces transcription of virulence factors that target human PMNs, making these strains ideal models for comparing cytotoxicity using the assays described. Tailoring the growth conditions, volumes of supernatants added, or ratio of bacteria to PMNs may be required when using other strains of Staph aureus.
