Nov 21, 2020

Public workspacePMN- 06 - Culture of Human PMN - TNF-α production V.2

DOI
https://dx.doi.org/10.17504/protocols.io.bpx2mpqe
  • 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)
Farmacologia Medica
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DOI: https://dx.doi.org/10.17504/protocols.io.bpx2mpqe
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 06 - Culture of Human PMN - TNF-α production. protocols.io https://dx.doi.org/10.17504/protocols.io.bpx2mpqeVersion 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 21, 2020
Last Modified: November 21, 2020
Protocol Integer ID: 44762
Keywords: constituent cannabidiol inhibit human polymorphonuclear leukocyte function, culture of human pmn, human pmn
Abstract
Published work using this protocol:


- A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Mabou Tagne A, Marino F, Legnaro M, Luini A, Pacchetti B, Cosentino M.Int J Mol Sci. 2019 Apr 13;20(8):1833. doi: 10.3390/ijms20081833
Materials
MATERIALS
ReagentFetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentLipopolysaccharides from Escherichia coli O127:B8Merck MilliporeSigma (Sigma-Aldrich)Catalog # L3137
ReagentPenicillin/StreptomycinEuroCloneCatalog #ECB3001D - 100 ml
ReagentELISA kit for TNF-αThermo Fisher ScientificCatalog #KHC0031
ReagentCannabidiolLinnea SACatalog #not available
Instrumentation needed:

Sterile and non sterile plastic disposables
ELISA plate reader
Troubleshooting
Isolate PMN according to the protocol PMN-01a or PMN-01b.
Resuspend PMN at 10x106cells/ml in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin (SOLUTION- 13).


Document
NAME

SOLUTION- 13 - Complete culture medium

CREATED BY
Farmacologia Medica

Add Amount300 µL of cell suspension in a 5 ml tube for cell culture and incubate for Duration21:00:00 at Temperature37 °C in 5% CO2. Cells were incubated in resting condition or in presence of stimulus. Stimulus (LPS 1 µg/ml and/or CBD 10-8-10-5M) in 5 ml tubes for cell culture. Other possible activating stimuli can be selected and included.

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 (Amount150 µL x2 aliquots) and cell pellet.

Store the supernatant at -80°C until the TNF-α Enzyme Linked-Immuno-Sorbent Assay (ELISA) is performed.
Store cell pellet at -80°C for gene expression analysis (RT PCR).