Nov 21, 2020
PMN- 01a - Isolation of Human PMN from Buffy Coat
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano Legnaro1,
- Emanuela Rasini1,
- Marco Ferrari1,
- Franca Marino1
- 1Center for Research in Medical Pharmacology, University of Insubria (Varese)
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 01a - Isolation of Human PMN from Buffy Coat. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxxmppn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 21, 2020
Last Modified: November 21, 2020
Protocol Integer ID: 44759
Keywords: constituent cannabidiol inhibit human polymorphonuclear leukocyte function, buffy coat separation of human neutrophil, human neutrophil, isolation of human pmn, human polymorphonuclear leukocyte, granulocytes from human blood, production by human polymorphonuclear leukocyte, granulocyte, mononuclear cell, human pmn
Abstract
Separation of Human Neutrophils (PMN) from Buffy Coat: list of published papers using this protocol
- Boydum A.Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Lab. Invest. 21 (Suppl.97): 77-89, 1968
- 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.
- 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.
Materials
- Ethylenediaminetetraacetic acid disodium salt dihydrate: Sigma AldrichCatalog #ED2SS
- Ficoll Paque PLUS: Ge HealthcareCatalog #17144003-500 ml
- Fetal Bovine Serum (FBS): EuroCloneCatalog #ECS0180L-500 ml
-RPMI 1640: EuroCloneCatalog #ECM 0495L- 500 ml
- NaCl: Sigma AldrichCatalog #S9625
- NH4Cl: Merck Serono GmbHCatalog #1.01145.1000
- KHCO3: Merck Serono GmbHCatalog #1.04854.500
- Acetic Acid 100%: Sigma AldrichCatalog #A6283
- Genitian violet 1%: Marco VitiCatalog #not available
Optical Microscope
Troubleshooting
Before start
All reagents used in this protocol must be at room temperature
Place 5 mL of venus blood from BUFFY COAT into 10 ml volume centrifuge tube.
Add 2 mL of SOLUTION- 03 and mix well drawing in and out of a pipette.
Incubate in the DARK for 00:45:00 at 37 °C
45m
Place 3 mL of Fycoll-HyPaque media solution into a 10 ml volume centrifuge tube.
Slowly and carefully layer the supernatant from blood/dextran mixture onto the Fycoll-HyPaque media solution.
Note
Important: when layering the sample, do not mix the Fycoll-HyPlaque media solution and supernatant
Centrifuge at 400 x g, Room temperature, 00:30:00 , no break
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
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 5 mL of NaCl 0.15 Molarity (M) (SOLUTION- 01)and centrifuge at 400 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Aspirate the supernatant with a plastic Pasteur pipette.
Lyse remaining red blood cells in 5 mL of hypotonic Lysis Buffer (SOLUTION- 06) for 00:05:00
5m
Centrifuge at 400 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Aspirate the supernatant with a plastic Pasteur pipette.
Resuspend the pellet in 5 mL NaCl 0.15 Molarity (M) (SOLUTION- 01)
Centrifuge at 400 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Aspirate the supernatant with a plastic Pasteur pipette.
Resuspend the cell pellet in 5 mL NaCl 0.15 Molarity (M) (SOLUTION- 01)for manula cell counting with Türk solution (follow protocol CELL COUNT- 02).
OPTIONAL STEP
Cell viability can be checked using protocol CELL COUNT- 03 in the automated cell counter with Trypan Blue solution.
Equipment
Cellometer Auto T4
NAME
Automated Cell Counter
TYPE
Nexcelom Bioscience
BRAND
Euroclone
SKU
OPTIONAL STEP
If needed, check the purity of PMN suspension by using morphological parameters of the flow cytometer.
For this test 0,5 x106 PMN in 500 µL of PBS (SOLUTION- 02) are enough.
Equipment
BD FACS Celesta
NAME
Flow Cytometer
TYPE
Becton Dickinson
BRAND
Milan Italy BD
SKU
EXPECTED RESULTS
Expected result
VIABILITY: the expeted viability by Tripan Blue shuold be ≥90%
CELL YIELD: ± 6 x106 cells starting from 1 mL of Buffy Coat