TITLE: PMN- 01a - Isolation of Human PMN from Buffy Coat
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, 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 venus blood from BUFFY COAT into 10 ml volume centrifuge tube.
5 mL
?. Add  of SOLUTION- 03 and mix well 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-HyPlaque media solution and supernatant
?. Centrifuge at  , no break
Centrifuge: 400 34, 30 min
?. 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 Molarity (M) (SOLUTION- 01)and centrifuge at
5 mL
Centrifuge: 400 34, 5 min
?. Aspirate the supernatant with a plastic Pasteur pipette.
?. Lyse remaining red blood cells in  of hypotonic Lysis Buffer (SOLUTION- 06) for
5 mL
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?. Centrifuge at
Centrifuge: 400 34, 5 min
?. Aspirate the supernatant with a plastic Pasteur pipette.
?. Resuspend the pellet in NaCl 0.15 Molarity (M) (SOLUTION- 01)
5 mL
?. Centrifuge at
Centrifuge: 400 34, 5 min
?. Aspirate the supernatant with a plastic Pasteur pipette.
?. Resuspend the cell pellet in NaCl 0.15 Molarity (M) (SOLUTION- 01)for manula cell counting with Türk solution (follow protocol CELL COUNT- 02).
5 mL
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?. OPTIONAL STEPCell viability can be checked using protocol CELL COUNT- 03 in the automated cell counter  with Trypan Blue solution.
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?. OPTIONAL STEPIf needed, check the purity of PMN suspension by using morphological parameters of the flow cytometer.For this test 0,5 x106  PMN in of PBS (SOLUTION- 02) are enough.
500 µl
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?. EXPECTED RESULTS
VIABILITY:  the expeted viability by Tripan Blue shuold be ≥90%CELL YIELD: ± 6 x106 cells starting from 1 mL of Buffy Coat