Jul 07, 2020
PMN- 05 Culture of Human PMN - IL-8 production
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano LM 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 LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 05 Culture of Human PMN - IL-8 production. protocols.io https://dx.doi.org/10.17504/protocols.io.bhdgj23w
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: June 10, 2020
Last Modified: July 07, 2020
Protocol Integer ID: 38024
Abstract
Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes.
Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.
Materials
MATERIALS
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Lipopolysaccharides from Escherichia coli O127:B8SigmaCatalog # L3137
N-FORMYL-MET-LEU-PHE (fMLP)Sigma AldrichCatalog #F3506
ELISA Kit for IL-8InvitrogenCatalog # KHC0081
Instrumentation needed
Sterile and not sterile plastic disposable
Laminar flow hood
ELISA plate reader
Isolate PMN according either to the protocol "Separation of Human Neutrophils (PMN) from Buffy CoatV.2" or "Separation of Human Neutrophils (PMN) from Whole Blood".
Resuspend PMN at 10x106 cells/ml in RPMI medium.
Use a number of tubes according to the experimental plan and to the substances to test.
Tubes will be prepared to test resting IL-8 production (substances alone) and stimulus-induced production.
Note
Add substances before the activation with stimulus (time selected according to the experiental plan)
Stimulus: fMLP 0.1 μM or LPS 1 μM or other.
Incubate PMN at 37 °C for 03:00:00 in a 5 ml tubes for cell culture. Cells will be incubated in resting conditions or in presence of stimulus (point 3-4).
Centrifuge at 1500 x g, 00:10:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Harvest cell-free supernatant using a pipette and cell pellet (for real-time assay of mRNA for IL-8).
Store the supernatant and cell pellet at -80°C until the IL-8 Enzyme Linked-ImmunoSorbent Assay (ELISA) and/or real time assay is performed.