Jul 07, 2020

Public workspacePMN- 04 Measurement of Intracellular Reactive Oxygen Species (ROS) levels in Human PMN

DOI
dx.doi.org/10.17504/protocols.io.bhc9j2z6
  • 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy);
  • 2Center for Research in Medical Pharmacology, University of Insubria (Varese , Italy)
Mattia Di Rocco
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DOI: dx.doi.org/10.17504/protocols.io.bhc9j2z6
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 04 Measurement of Intracellular Reactive Oxygen Species (ROS) levels in Human PMN. protocols.io https://dx.doi.org/10.17504/protocols.io.bhc9j2z6
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: 38017
Abstract
List of published work using this protocol:

  • Immune Profile of Hyaluronic Acid Hydrogel Polyethylene Glycol Crosslinked: An in Vitro Evaluation in Human Polymorphonuclear Leukocytes. Franca Marino, Marco Cosentino, Massimiliano Legnaro, Alessandra Luini, Julia Sigova, Roberto Mocchi, Trello Lotti, Nicola Zerbinati. Dermatol Ther. 2020 Apr 10; e13388. doi: 10.1111/dth.13388. Online ahead of print.

  • A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti, Marco Cosentino. Int J Mol Sci. 2019 Apr 13;20(8):1833. doi: 10.3390/ijms20081833.

  • β 2 -Adrenoceptors Inhibit Neutrophil Extracellular Traps in Human Polymorphonuclear Leukocytes. Franca Marino, Angela Scanzano, Laura Pulze, Monica Pinoli, Emanuela Rasini, Alessandra Luini, Raffaella Bombelli, Massimiliano Legnaro, Magda de Eguileor, Marco Cosentino . J Leukoc Biol. 2018 Sep;104(3):603-614. doi: 10.1002/JLB.3A1017-398RR. Epub 2018 Apr 18.

  • 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.

  • The Essential Oil of Bergamot Stimulates Reactive Oxygen Species Production in Human Polymorphonuclear Leukocytes. Marco Cosentino, Alessandra Luini, Raffaella Bombelli, Maria T Corasaniti, Giacinto Bagetta, Franca Marino. Phytother Res. 2014 Aug;28(8):1232-9.doi: 10.1002/ptr.5121. Epub 2014 Jan 23.

  • Immunomodulatory Properties of Achyrocline Satureioides (Lam.) D.C. Infusion: A Study on Human Leukocytes. Marco Cosentino, Raffaella Bombelli, Elena Carcano, Alessandra Luini, Franca Marino, Francesca Crema, Federico Dajas, Sergio Lecchini. . J Ethnopharmacol. 2008 Mar 28;116(3):501-7. doi: 10.1016/j.jep.2007.12.014. Epub 2008 Jan 6.

Materials
MATERIALS
ReagentNaClSigma AldrichCatalog #S9625
ReagentC-DCFH-DAInvitrogen - Thermo FisherCatalog #C2938
ReagentKClSigmaCatalog #P9541
ReagentMgSO4SigmaCatalog #1.05886
ReagentGlucose SigmaCatalog #1.08337
ReagentCaCl2SigmaCatalog #1.02382
ReagentHepesSigmaCatalog #H7006
Dubnoff Bath


Suspend isolated PMN at 1x106 cells/ml in HBSS/HEPES medium.

Document
HBSS/HEPES for ROS measurement in PMN
NAME

HBSS/HEPES for ROS measurement in PMN

CREATED BY
Mattia Di Rocco

Add 2x106 cells in Amount2 mL of HBSS/HEPES for each tube.

Incubate forDuration01:00:00 with Concentration2 micromolar (µM) of C-DCFH-DA in the dark at Temperature37 °C (Dubnoff Bath).




Wash twice with Amount2 mL of HBSS/HEPES.
Note
Each time centrifuge at Centrifigation400 x g, Room temperature, 00:05:00







Equipment
Centrifuge 5804
NAME
Eppendorf
BRAND
Eppendorf
SKU




Aspirate supernatant and resuspend with Amount2 mL of HBSS/HEPES.

Recover PMN in cuvette of 2 ml in the dark at Temperature37 °C (Dubnoff Bath)

Measurements of fluorescence must be performed by means of a Spectrofluorimeter with excitation wavelength of 488 nm.

Equipment
LS 50B
NAME
Spectrofluorimeter
TYPE
Perkin Elmer
BRAND
Perkin Elmer instruments
SKU



Cuvette must be maintained at Temperature37 °C by means of a thermostatic bath, that will be turned on before the starting ROS measurement.

Critical
Fluorescence emission must be collected at 525 nm and recorded in a specific data table that will be signed for each specific experimental plan (time for recording and complete time analysis), that will be signed by the researcher.
Intracellular ROS levels must be expressed as fluorescence intensity, in arbitrary units (AU) or according to the specific experimental plan.