Add 100 microliters of the NET stock into each well of a 96-well flat bottom plate, and incubate the plate overnight at four degrees Celsius in the dark to coat the wells. 12 to 20 hours later, use a microscope to verify formation of a uniform monolayer of cell-free NETs at the bottom of the wells. Once the monolayer has been verified, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the monolayer at the bottom.
This is the most challenging aspect of this procedure. In order to maintain an adequate NET monolayer, you have to handle the plate very carefully. Add all reagents and cells slowly on the side of the wells. Aspirate very gently, and avoid touching the bottom of the well with the suction tip.
With the non-adherent material removed, gently add 100 microliters of a 1% bovine serum albumin blocking solution to each well, and leave for one hour at room temperature. Do not agitate or shake the plate as this can disrupt the NET monolayer.
Next, prepare A549 cancer cells by harvesting them when they are 70 to 80% confluent using standard techniques. Resuspend the cells in medium at a concentration of 2 x 104 cells per 100 microliters. Stain the cells by adding one microliter of CFSE per milliliter of medium, and leave them at room temperature for 10 minutes.
Next, centrifuge the cells at 450 times g at four degrees Celsius for five minutes. Discard the supernatant, and resuspend the cells in the initial volume of medium to maintain a concentration of 2 x 104 cancer cells per 100 microliters of medium.
Take the NET-coated plate, and gently aspirate the blocking solution. Then, add 100 microliters of the cancer cell suspension to each well, and allow the cells to adhere to the plate for 90 minutes at 37 degrees Celsius and 5% carbon dioxide.
Fully aspirate the liquid, and add 1,000 units of DNase I to some wells for 10 minutes to degrade the NETs. In other wells, add 100 microliters of sterile water per well for 10 minutes as a vehicle control. Next, gently aspirate the liquid from each well, and wash with 100 microliters of PBS to remove any non-adherent A549 cells.
Aspirate and discard all solution in the wells, leaving only the NETs and adherent cancer cells at the bottom. Then, add 100 microliters of a 4% formaldehyde solution per well to fix the cells. Immediately transfer the plate to a fluorescence microscope to read the assay, and then, plot and analyze the results using the data analysis software.
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