Nov 21, 2020

Public workspacePMN- 05 Culture of Human PMN - IL-8 production

DOI
dx.doi.org/10.17504/protocols.io.bpx3mpqn
  • 1Center for Research in Medical Pharmacology, University of Insubria (Varese)
Farmacologia Medica
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DOI: dx.doi.org/10.17504/protocols.io.bpx3mpqn
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.bpx3mpqn
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: 44763
Abstract
Published work using this protocol:

- 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 1640: EuroCloneCatalog #ECM 0495L- 500 ml
- Lipopolysaccharides from Escherichia coli O127:B8: SigmaCatalog # L3137
- N-FORMYL-MET-LEU-PHE (fMLP): Sigma AldrichCatalog #F3506
- ELISA Kit for IL-8: InvitrogenCatalog # KHC0081



Instrumentation needed

- Sterile and not sterile plastic disposable
- Laminar flow hood
- ELISA plate reader
Isolate PMN according either to the protocol "PMN- 01a" or "PMN- 01b".
Resuspend PMN at 10x106 cells/ml in complete culture medium (SOLUTION- 13).


Document
SOLUTION- 13 - Complete culture medium
NAME

SOLUTION- 13 - Complete culture medium

CREATED BY
Farmacologia Medica

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 atTemperature37 °C forDuration03:00:00 in a 5 ml tubes for cell culture. Cells will be incubated in resting conditions or in presence of stimulus (point 3-4).

3h
Centrifuge at Centrifigation1500 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.