Neutrophils are terminally differentiated cells, and currently there are no cell lines to fully recapitulate neutrophil biology. Thus it is necessary to obtain pure, inactivated, healthy, and fresh neutrophils to study their biology. This refresh method combines density gradient, sedimentation, gentle lysis to obtain a pure neutrophilic preparation.
It's easy to set up, requires simple equipment and little practice. The technical aspect, such as gradient layering, of this method will require some practice to master, but once the gradient is perfected the other steps should come as a breeze. Begin by sterilizing the buffy coat, packaging, and laminar hood.
Then add 10 milliliters of the blood into a 50 milliliter tube. To dilute the blood for a cleaner of gradient, add 5%FBS/HBSS and makeup the volume up to 35 milliliters. After closing the lid, invert the tube several times for mixing and then keep the tube upside down to obtain the bottom devoid of red blood cells.
Add 10 milliliters of the density gradient medium directly beneath the blood, ensuring that the medium and the blood do not mix and the interface remains sharp. Spin the tube at 400 times g for 30 minutes at room temperature, making sure to disable the brake. After spinning, observe the gradient separated into a top serum plasma layer, a middle white ring of peripheral blood mononuclear cells, a cloudy density gradient medium layer, and a bottom pellet consisting of a white thin neutrophil band on top of the red blood cells.
To remove PBMC, emerge the suction pipette directly into the PBMC layer and aspirate completely while the serum plasma layer is getting decreased as the ring is removed. Scrape the side of the tube with the suction pipette to maximize the removal of the PBMC. Carefully remove the cloudy density gradient medium layer between the PBMC ring and neutrophil/RBC pellet.
For erythrocyte sedimentation, use a 10 milliliter pipette to transfer the neutrophil/RBC pellet into a clean tube. Then add 5%FBS/HBSS to a final volume of 25 milliliters. Directly add 25 milliliters of the prewarmed solution containing 3%dextran/0.9%NaCl in water into the tube and mix gently by inversion.
Place the tube on a leveled and non-vibrating surface for 15 minutes. After placing the tube back into the hood, slightly immerse the pipette in the liquid and collect approximately 30 milliliters of the top layer following the liquid surface downwards. Spin the tube to obtain a red pellet without floating particles in the media.
For lysis of the residual RBC, gently aspirate the supernatant without disrupting the pellet. Add 25 millimeters of sterile ultrapure water directly into the tube and gently mix by inverting the tube for 28 seconds to lyse the RBC. Then, immediately add 25 milliliters of sterile 1.8%NaCl solution prepared in water into the tube and bring the solution back to isotonic conditions by gentle mixing.
Spin the tube at 200 times g for three to five minutes with a low brake to minimize the sedimentation of RBC and platelets with neutrophils. For resuspending the white neutrophil pellet, directly add the culture medium on the pellet but do not pipette up and down. Next, rock the tube horizontally from side to side to minimize cell activation.
If cell aggregation or clumping is observed, filter the cell suspension through a 70 micrometer mesh to discard the clumped neutrophils. For assessing the quality of the isolated neutrophil preparation, stain the cells with markers specific for neutrophils, eosinophils, and an activation marker. After acquiring 20, 000 cells by flow cytometry, analyze the cell purity and activation using the gating strategies and determine the cell viability using Annexin V/propidium iodide as described in the text manuscript.
The density gradient with a low speed yielded neutrophils with more purity, while a high speed resulted in increased yield at the expense of purity. Using the fluorescence-activated cell sorting, the cell distribution alone provided an estimate of the cell isolation quality, but the use of specific cell markers should be preferred. The contaminating cell populations identified were monocytes, lymphocytes, and eosinophils.
The enormous neutrophil yield was achieved using this protocol. The expression of CD62L was assessed. The mean fluorescent intensity for CD62L was decreased in the positive control cells, indicating CD62L shedding and neutrophil activation.
The neutrophil health should be assessed before performing the assay as the neutrophils have a relatively short half-life and activation further shortens the life. After neutrophil purification using the density gradient and commercial microbeads, the cells were cultured for 24 hours and cell survival was analyzed by flow cytometry. The quantification of viable cells indicated that the density gradient purification resulted in more viable cells after 24 hours than purification using a kit.
It is important that the gradient layering and centrifugation steps be performed as well as possible as it will greatly impact the quality of the preparation. In vitro experiments and biochemical assays can be performed following this procedure. Also, negative selection could be performed if ultrapure cells are required as in cytokine and protein expression experiments.
