Our research aims to understand the role of neutrophil subsets and how they contribute to the pathogenesis of inflammatory diseases such as lupus. In a broader sense, our work also demonstrates the heterogeneity of neutrophil populations in human biology and how this can change based on varying physiological states. Emerging research shows that neutrophil populations in humans are heterogeneous.
However, neutrophils continue to be studied as a single population due to difficulty in isolating subtypes reliably without activation and in sufficient quantity. Our protocol provides a method for untouched isolation and basic characterization of neutrophil subtypes, overcoming these problems. Recent research, though limited, has shown that neutrophil subtypes, such as low-density neutrophils, play a role in altered physiological states such as pregnancy and inflammation as well as in diseases like tuberculosis, autoimmunity, and cancer.
Our protocol will enable reproducible, robust research into the contributions of these cells to human health and disease. Now that we have a tested robust protocol for separating low-density neutrophils from normal density neutrophils, we are focusing on answering basic questions pertaining to how these low-density neutrophils present in inflammatory conditions. We are especially interested in how these are different from normal density neutrophils in their cell numbers, immuno phenotype, immuno metabolism, and more.
To begin, label four 50 milliliter conical tubes for isotonic working solutions of 100%81%70%and 55%Add 27 milliliters of density gradient medium to the first tube labeled 100%Add 3 milliliters of 10X PBS. Next, measure out the required volumes of isotonic 100%working solutions and 1X PBS for each concentration of the working solutions. Add it to the respective conical tubes.
Use a Pasteur pipette to mix thoroughly for homogeneity. Now carefully layer 3 milliliters of the isotonic 81%working solution at the bottom of a 15-milliliter conical tube. Then slowly and gently layer 3 milliliters of the isotonic 70%working solution on top, ensuring that the layers do not mix.
To prepare the cell separation buffer, combine 2%fetal bovine serum, 1 millimole EDTA and PBS to a total volume of 500 milliliters. Pipette to mix thoroughly for uniformity. Vortex the magnetic beads thoroughly for 30 seconds to ensure they are fully resuspended before use.
For each donor, aliquot 4 milliliters of whole blood into three separate 14-milliliter round bottom tubes. Pipette 200 microliters of neutrophil isolation cocktail and 200 microliters of magnetic beads into each tube. Gently pipette the solution to resuspend the mixture before incubating to allow the reagents to bind to unwanted cells.
Next, add the cell separation buffer to each tube to make up the volume to 12 milliliters and mix well. Place the tubes without lids on a magnetic rack and incubate. Now use a serological pipette to carefully transfer the clear cell suspension containing neutrophils from each tube into a new clean tube.
Then remove the original tube from the magnet. Vortex the magnetic beads again for 30 seconds. Add 200 microliters of the magnetic beads to the newly transferred cell suspension before resuspension and incubation as demonstrated earlier.
Place the tubes back onto the magnet without lids. After a 10-minute incubation at room temperature, transfer the clear cell suspension containing isolated neutrophils into a new tube. Pull the isolated neutrophil suspensions obtained from whole blood into a single 50-milliliter tube.
Top up the tube to a total volume of 50 milliliters using the cell separation buffer. Centrifuge with the break on, then discard the supernatant and resuspend the neutrophil pellet in 1 milliliter of cell separation buffer. After centrifuging the suspension again, discard the supernatant and resuspend the neutrophil pellet in 3 milliliters of the 55%density gradient medium.
Slowly layer the 3 milliliters of 55%density gradient medium containing the cell suspension onto the pre-made density gradient tubes, avoiding disturbance of the gradient. Centrifuge the tubes at 720 G for 30 minutes without using the break. Following centrifugation, handle the tubes carefully to avoid disturbing the layers.
With a transfer pipette, carefully isolate the separate layers containing low and normal density neutrophils and transfer them to separate labeled 15-milliliter tubes. To wash off any residual density gradient medium, add PBS to each 15-milliliter tube up to the 15-milliliter mark. Next, centrifuge at 400 G for 5 minutes at room temperature with the break on.
Count the cells in each fraction on a hemocytometer before further experimentation. The layering of total neutrophils over the density gradient medium resulted in the successful formation of two distinct bands. With the low-density neutrophils forming an upper band and the normal density neutrophils forming a lower band.
Overloading the density gradient medium with neutrophils above 5-6 million cells per milliliter caused the bands to appear diffuse, increasing the risk of subtype mixing. The average isolation purity was 93.8%for low-density neutrophils, and 96.3%for normal density neutrophils with minimal contamination from other cell types.
