Nov 22, 2020
Version 2
PMN- 01b - Isolation of Human PMN from Whole Blood V.2
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano Legnaro1,
- Emanuela Rasini1,
- Marco Ferrari1,
- Franca Marino1
- 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy)
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 01b - Isolation of Human PMN from Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxymppwVersion created by Farmacologia Medica
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 22, 2020
Last Modified: November 22, 2020
Protocol Integer ID: 44760
Keywords: constituent cannabidiol inhibit human polymorphonuclear leukocyte function, isolation of human pmn, human polymorphonuclear leukocyte, production by human polymorphonuclear leukocyte, granulocytes from human blood, mononuclear cell, human pmn, granulocyte
Abstract
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
MATERIALS
Ficoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
KHCO3MerckCatalog #1.04854.500
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED2SS
Acetic Acid 100%Merck MilliporeSigma (Sigma-Aldrich)Catalog #A6283
Gentian violet 1%Marco VitiCatalog #not available
NH4ClMerckCatalog #1.01145.1000
Optical Microscope (for manual cell count)
Troubleshooting
Before start
All reagents used in this protocol must be at room temperature
Place 5 mL of whole blood into a 10 ml volume centrifuge tube.
Add 2 mL of dextran solution (SOLUTION- 03) and mix well by drawing in and out of a pipette.
Incubate in the dark for 00:30:00 at 37 °C
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-HyPaque media solution and supernatant.
Centrifuge at 400 x g, Room temperature, 00:30:00 with no brake.
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 µL of NaCl 0.15 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 pipette Pasteur.
Lyse remaining red blood cells in 5 mL of hypotonic Lysis Buffer (SOLUTION- 06) for 00:05:00
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 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 M (SOLUOTIN- 01) for manual cell counting using Türk solution (SOLUTION- 08). Follow protocol CELL COUNT- 02.
OPTIONAL STEP
If needed, check the purity of PMN suspension by using morphological parameters of the flow cytometer.
For this test 0,5x106 PMN in 500 µL of PBS (SOLUTION- 02) are enough.
Equipment
BD FACSCelesta
NAME
Flow Cytometer
TYPE
Becton Dickinson, Milan Italy
BRAND
BD
SKU
EXPECTED RESULTS
Expected result
VIABILITY: the expeted viability by Trypan Blue shuold be ≥90%
CELL YIELD: ± 1 x106 cells starting from 1 mL of Whole Blood