The overall goal of the following experiment is to demonstrate a simple protocol for isolating neutrophil, extracellular traps or nets from human whole blood using readily available reagents. This is achieved by first isolating neutrophils from human whole blood, using a differential centrifugation technique as a second step. Isolated neutrophils are stimulated with PMA to induce net formation, also called NETosis.
Next, a plate is prepared with a cell-free net isolate and cancer cells are added to demonstrate how the nets affect adhesion of the cells. The results show that the addition of a net monolayer improves adhesion of a 5 49 cancer cells and that this adhesion is reduced when the nets are degraded. Using DNAS one Visual demonstration of this method is critical.
Given the novelty of this technique handling of neutrophil xor cellular traps are nets extremely difficult due to how fragile these structures are. To begin, obtain 80 milliliters of fresh human whole blood in 14 heparinized green top tubes as described in the text protocol. Open each tube and add five milliliters of PBS without calcium and magnesium to each tube.
Next, add 15 milliliters of lymphocyte separation media or LSM into each of four 50 milliliter conical tubes at room temperature. Then use an 18 gauge needle mounted on a 60 milliliter syringe to carefully layer the diluted blood onto the LSM, creating a sharp LSM blood interface centrifuge the tubes at 800 times G for 30 minutes at 21 degrees Celsius without breaking to stop the centrifuge carefully aspirate and discard the top two liquid layers and the interface between them leaving only the bottom red layer. This pellet contains erythrocytes and neutrophils.
Add 20 milliliters of PBS and 20 milliliters of 6%dextran solution to each tube. Gently invert each tube and allow them to sit at room temperature for 30 minutes. The red blood cells will sediment to the bottom of the tube.
Next, transfer the supernatants into fresh tubes and discard the pallets. Centrifuge the supernatants at 450 times G at four degrees Celsius for five minutes, and then discard the supernatant, leaving the neutrophil rich pellet. Prepare a lysing solution with 0.5 milliliters of lysis buffer in 4.5 milliliters of sterile water.
Add a total of five milliliters of the lysing solution to each tube and pool all of the pellets into one flask. Place the flask in the dark for 10 minutes at room temperature to lice any remaining red blood cells. Centrifuge the neutrophils at 450 times G at four degrees Celsius for five minutes.
Discard the supernatant and wash the pellet with five milliliters of PBS without calcium and magnesium. Then centrifuge the sample again at 450 times G at four degrees Celsius and remove the supernatant containing the remaining lysis solution. Resuspend the neutrophil pellet in 30 milliliters of cold RPMI medium supplemented with 3%fetal bovine serum, and put the tube on ice to stimulate the formation of neutrophil extracellular traps or nets.
Add 500 nano molar of PMA to 30 liters of the neutrophil suspension in a 150 millimeter by 25 millimeter flat tissue culture dish. With a 20 millimeter grid, incubate the cells for four hours at 37 degrees Celsius under 5%carbon dioxide. After incubation, gently aspirate and discard the medium, taking care not to disrupt the layer of nets and neutrophils adhere to the bottom of the dish.
Then use a pipette to wash the bottom of each dish with 15 milliliters of cold PBS without calcium and magnesium, all adherent material should be removed from the bottom. Collect the wash suspension in a 15 milliliter conical tube and then centrifuge at 450 times G for 10 minutes. At four degrees Celsius neutrophils and any remaining cells will pellet at the bottom.
Leaving a cell free net rich supernatant next decant the supernatant into multiple 1.5 milliliter tubes and centrifuge at eight 18, 000 times G for 10 minutes at four degrees Celsius. To pellet the DNA discard the supernatant and resuspend the pellet in PBS such that the concentration corresponds to two times 10 to the seventh neutrophils per 100 microliters of PBS. This cell-free net stock can be used for subsequent experiments.
Measure the DNA concentration of the sample using spectro photometry. An adequate concentration should range between 140 and 180 nanograms per microliter at 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 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 a 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 mono layer.
Next, prepare a 5 49 cancer cells by harvesting them when they are 70 to 80%confluent. Using standard techniques, re suspend the cells in medium at a concentration of two times 10 to the fourth cells per 100 microliters. Stain the cells by adding one microliter of CFS e 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 two times 10 to the fourth 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 1000 units of DNAs, one 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.
A 5 49 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.
Cell-free nets were used in static adhesion assays with a 5 49 cancer cells. Light microscopy showed that cancer cells strongly adhere to the net monolayer. In vitro.
This adhesion decreased after the net monolayer was degraded by DNA one fluorescence microscopy of labeled a 5 49 cancer cells confirmed these results. Binding of a 5 49 cells to the net mono layer was quantified by counting the number of cells per high power field. As shown here, there was no change in cancer cell adhesion to nets in the vehicle control.
However, there was a significant decrease in cancer cell adhesion to nets in the presence of DNA one. After watching this video, you should have a good idea of how to isolate and handle nets from whole human blood. These cell-free net isolates can then be used in a variety of techniques, including immunofluorescence, confocal microscopy, electron microscopy, and western blotting.
