Nov 21, 2020
Version 2
PMN- 02 - MTT Assay in Human PMN V.2
- 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- 02 - MTT Assay in Human PMN. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxzmpp6Version 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: 44761
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
Dimethyl sulfoxide (DMSO)Sigma AldrichCatalog #276855
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Thiazolyl blue tetrazolium bromide (MTT)Sigma AldrichCatalog #M5655
Penicillin/StreptomycinEuroCloneCatalog #ECB3001D - 100 ml
Instrumentation needed:
Sterile and non sterile plastic disposables
ELISA plate reader
Before start
Use all reagents at Room Temeperature
Isolate PMN according to the protocol PMN-01a for buffy coats or PMN-01b for fresh whole blood.
Resuspend PMN at 1x106cells/ml in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (SOLUTION- 13).
Document
NAME
SOLUTION- 13 - Complete culture medium
CREATED BY
Farmacologia Medica
Add 250 µL of cell suspension per well in a 96-well round bottom plate and incubate for 24:00:00 alone or in the presence of the test substance at 37 °C in a 5% CO2 atmosphere. Cells must be plated in duplicate for each experimental condition.
Centrifuge at 1400 x g, 00:05:00 and discard the supernatant.
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Resuspend cells with 200 µL of MTT solution at 0.5 mg/ml, incubate for 02:00:00 at 37 °C in a 5% CO2 atmosphere.
Centrifuge at 1400 x g, 00:05:00 and discard the supernatant.
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Add 120 µL of DMSO per well, mix the plate until the formazan crystals are dissolved.
Read the plate on the ELISA plate reader at 570 nm.