TITLE: Separation of Human Neutrophils (PMN) from Whole Blood
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Emanuela Rasini, Massimiliano Legnaro, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Separation of Human Neutrophils (PMN) from Buffy Coat: list of published papers using this protocol</div><div class = "text-block">- Boydum A.Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Lab. Invest. 21 (Suppl.97): 77-89, 1968</div><div class = "text-block">- Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti and Marco Cosentino. A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Int J Mol Sci2019 Apr; 20(8): 1833. Published online 2019 Apr 13. doi: 10.3390/ijms20081833.</div><div class = "text-block">- Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.</div></div>

[STEPS]
?. Place  of whole blood into a 10 ml volume centrifuge tube.
5 mL
?. Add  of dextran solution and mix well by drawing in and out of a pipette.
2 mL
?. Incubate in the dark for  at
37 °C
?. Place  of Fycoll-HyPaque media solution into a 10 ml volume centrifuge tube.
3 mL
?. Slowly and carefully layer the supernatant from blood/dextran mixture onto the Fycoll-HyPaque media solution.
Important: when layering the sample, do not mix the Fycoll-HyPaque media solution and supernatant.
?. Centrifuge at  with no brake.
Centrifuge: 400 34, 30 min, 20 10
?. Draw off the mononuclear cell layer at the Ficoll/plasma interface along with plasma and Ficoll media, leaving the white cell layer of granulocytes above the red blood cell layer undisturbed.
?. Resuspend the remaining cell layer in  of NaCl 0.15 M and centrifuge at
5 mL
Centrifuge: 400 34, 5 min, 20 10
?. Aspirate the supernatant with a plastic pipette Pasteur.
?. Lyse remaining red blood cells in of hypotonic lysis buffer for
5 mL
?. Centrifuge at
Centrifuge: 400 34, 5 min, 20 10
?. Aspirate the supernatant with a plastic Pasteur pipette.
?. Resuspend the pellet in  NaCl 0.15 M.
5 mL
?. Centrifuge at
Centrifuge: 400 34, 5 min, 20 10
?. Aspirate the supernatant with a plastic Pasteur pipette.
?. Resuspend the cell pellet in NaCl 0.15 M for cell counting.
5 mL
?. Mix  of cell suspention with an equal amount of Türk solution (diluition factor=2) allow mixture at Room temperature (RT).Take  of the mixture and place it inside a Bürker chamber and view under an optical microscope using 40x magnifiation Count cell in each square found in the four corners and in the central square (figure 1 below), including those that lie on the bottom and left-hand perimeters, but not those that lie on the top and right hand perimeters (see figure 2 below).Total number of cell per ml = mean number of cell x diluition factor x 104 (hemacitometer volume)Figure 1The gridded area of the chamber consists of nine 1 mmq squares. These squares are subdivided in three directions; 0.0625 mmq, 0.05 mmq and 0.04 mmq. The central square here in Figure 1 is further subdivided into 0.0025 mmq = 1/25 mmq squares. Count cells in 5 squares as shown.Figure 2Concerning those cells that lay on the perimeter of the square, count following this scheme.
10 µl
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10 µl
?. OPTIONAL STEPFor automatic cell count with Cellometer machines use Trypan Blue. The machine will calculate the number of cells /ml and the % of viability.Take   of cell suspension and add an equal amount of Trypan Blue. Use all the volume to place it in a counting chamber. Place the chamber inside Cellometer and count.
10 µl
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?. OPTIONAL STEPIf needed, check the purity of PMN suspension by using morphological parameters of the flow cytometer.For this test 0,5x106  PMN in   of PBS are enough.
500 µl
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?. EXPECTED RESULTS
VIABILITY:  the expeted viability by Tripan Blue shuold be ≥90%CELL YIELD: ± 1 x106 cells starting from 1 mL of Whole Blood