This protocol is significant because it allows for an in vitro analysis of neutrophil swarming, which overcomes many limitations of our present inability to experiment. The main advantage of this technique is that it provides easy access to the secreted cytokines and lipid mediators that neutrophils release during swarming and allows for quantitative imaging analysis. While we applied this technique to neutrophils, it could be extended to analyze the migration of other white blood cells like monocytes and T cells.
Begin by preparing a 1.6 milligram per milliliter cationic polyelectrolite, or CP solution, by adding the proper amount of CP powder to water and mixing it on the stir plate overnight or until all solids are dissolved. If desired, the solution can be made fluorescent by adding poly-L-lysine labeled with FITC. Generate the master silicon wafer using standard photolithography procedures.
The design used here is a four by four milliliter rectangular array of 30 micrometer diameter filled-in circles, with a 150 micrometer center to center spacing, but it can be modified as desired for different applications. Spin-coat a 40 micrometer thick layer of negative photoresist onto a silicon wafer. Then bake the wafer at 65 degrees Celsius for five minutes.
And 95 degrees Celsius for 10 minutes. Expose the wafer to UV light through a chrome photomask with 150 to 160 millijoules per centimeter squared. Bake the wafer again at 65 degrees Celsius for five minutes and 95 degrees Celsius for 10 minutes.
Then submerge it in photoresist developer for 10 minutes. And rinse it with isopropyl alcohol. The pattern should now be visible on the wafer.
Next, thoroughly mix a 10 to one ratio of polydimethylsiloxane, or PDMS, prepolymer and its curing agent. And pour the uncured mixture over the master wafer in a Petri dish. Vacuum treat the uncured PDMS mixture until no air bubbles are present.
Then cure it overnight at 65 degrees Celsius. On the next day, use a scalpel to cut around the exterior of the patterned section of the wafer. And slowly remove the cured PDMS slab, placing it on a clean cutting board with the patterned side facing up.
Punch out individual stamps from the PDMS slab with an eight millimeter biopsy punch and place each stamp face-down on the adhesive tape to remove debris. With these stamps facing up, prime each stamp with 100 microliters of the pre-prepared CP solution, ensuring that no air bubbles form between the solution and the stamp. Then invert the stamps onto a layer of CP solution and remove them after one hour.
Dab each wet stamp face down onto a clean glass slide six to eight times to remove excess liquid. Then vacuum treat the stamps for one to two minutes. Adhere an eight well, eight millimeter diameter imaging spacer on top of a clean glass slide as a guide for stamp placement.
Place a stamp face down on the glass slide in the center of each well of the imaging spacer. Then place a 5.6 gram balanced weight on top of each stamp and allow 10 minutes for stamping. Remove the weights and the stamps from the slide and allow the CP layer to dry at room temperature for 24 hours before adding bioparticles.
If the CP is tagged with FITC, check effectiveness of the stamping with a fluorescent microscope. Cut a blank PDMS slab to the size of the imaging spacer and use the eight millimeter biopsy punch to create wells in the PDMS that align with the wells of the spacer. Then adhere the PDMS slab to the glass slide.
Thaw a solution of bioparticles and dilute it to 500 micrograms per milliliter in water for injection. Add 100 microliters of bioparticle solution to each PDMS well on the glass slide and rock the slide for 30 minutes. Rinse the wells thoroughly with water and check the pattern on the slide with a fluorescent microscope.
The bioparticle microarray can be stored in a dust-free environment at four degrees Celsius for up to three months. Prepare the cells by resuspending them at 7.5 times 10 to the fifth cells per milliliter in IMDM with 0.4%human serum albumen. Add 100 microliters of the suspension to a PDMS well containing the bioparticle microarray, making sure that the cell suspension is convex over the top of the PDMS well and doesn't contain any bubbles.
Seal the well with a 12 millimeter diameter cover slip and press down gently with tweezers so the excess cell suspension escapes to edge of the well, then use a tissue to remove the excess. To image the cells, load the microparticle array with cells on the live cell imaging station of a microscope equipped with a cage incubator set to 37 degrees Celsius, 5%carbon dioxide, and 90%relative humidity. Use time lapse, fluorescent, and bright field microscopy to record images at 10X magnification every 10 seconds at 405 nanometers, 594 nanometers, and bright field.
Incubate the neutrophils in the wells with bioparticles at 37 degrees Celsius and 5%carbon dioxide for three hours, taking samples at desired time points. Use a bright field microscope to verify that swarms are formed on the microarray. Collect the entire volume of supernatant of a single well, and load it into a 0.45 micrometer centrifuge filter tube, making sure to analyze each time point in triplicate.
Centrifuge the tubes at 190 times G and 20 degrees Celsius for five minutes, and collect the filtered volume. Then store the samples at minus 80 degrees Celsius until processing time. When attempting this protocol, work with photoresist and developer in the fume hood.
Have an IRB approved protocol in place before taking blood from human donors. Remember that materials derived from human blood are BSL2. When neutrophils are added to the bioparticle microarray, neutrophils that contact the bioparticle clusters become activated and initiate the swarming response.
The bioparticle microarray was validated using time lapse fluorescent microscopy to track neutrophil migrations toward the bioparticle clusters. Stable neutrophil swarms are formed around each cluster after 30 to 60 minutes of exposure. In contrast, neutrophils do not show collective migration in the absence of bioparticle clusters.
Fluorescence intensity of stained neutrophil nuclei was used to determine that the average swarm size around the bioparticle clusters was approximately 1490 micrometers squared. In the absence of clusters, fluorescence of a given region of interest remained constant over time. Tracks of neutrophil migration show that neutrophils converged on a bioparticle cluster when one was present, but no convergence was observed in the control system.
The speed of swarming and nonactivated neutrophils was measured, and a statistically significant difference in the speed distributions was found. The average speed for swarming neutrophils was 20.6 micrometers per minute, while the average speed for control neutrophils was two micrometers per minute. The concentrations of 16 proteins that neutrophils release during swarming were analyzed over time.
10 proteins increased in concentration. Two decreased. And the remaining four increased during the first hour of swarming but decreased thereafter.
While performing this procedure, ensure that everything is clean and your stamps are dried properly, as these are essential for successful patterning of the bacterial particles. The development of this technique will enable researchers to explore new questions in the field of neutrophil swarming, including how free mediators and extracellular vesicles are involved in neutrophil intercellular communication.
