Nov 21, 2020
Version 2
PBMC- 05 - In Vitro Culture of TEFF+TREG - Cytokine Production by TEFF 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. PBMC- 05 - In Vitro Culture of TEFF+TREG - Cytokine Production by TEFF. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxwmppeVersion 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: 44758
Abstract
List of published work using this procedure:
- Kustrimovic, N., Comi, C., Magistrelli, L., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Minafra, B., Riboldazzi, G., Sturchio, A., Mauri, M., Bono, G., Marino, F., & Cosentino, M. (2018). Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients. Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8
Materials
MATERIALS
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Penicillin/StreptomycinEuroCloneCatalog #ECB3001D - 100 ml
L-Glutamine 100X - 100mLEuroCloneCatalog #ECB3000D
M-Phytohaemagglutinin powder Sigma AldrichCatalog #L8902-25 mg
Human Interleukin 2 lyophilized powder research grade Miltenyi BiotecCatalog #130-097-742
Instrumentation needed:
Sterile plastic disposables
Laminar Flow Hood
Humidified 37°C, 5% CO2 incubator
Isolate TEFF and TREG with Miltenyi Kit according to the protocol PBMC- 03.
Count both TEFF and TREG following the appropriate protocol (CELL COUNT- 02 or CELL COUNT- 03).
Use sterile 96-well round bottom plates.
Note
[Consider that these plates can contain a volume of maximum 250µL]
Centrifuge TEFF and TREG at 1200 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Resuspend TEFF and TREG in SOLUTION- 13 at a concentration of 1x106/mL.
Document
NAME
SOLUTION- 13 - Complete culture medium
CREATED BY
Farmacologia Medica
According to the experimental design, activate a desired number of wells containing TEFF cells with PHA 5µg/ml (final concentration) and IL-2 40 ng/mL (final concentration) by diluting the stock aliquots. Leave also wells of TEFF unstimulated (resting control).
Put TEFF-CPD labeled cells and TREG cells in the 96-well plate at a ratio of 1:1(for example, 0.1x106 TEFF + 0.1x106 TREG) and activate the cells il the well directly (see step 7 for concentrations): include 1 control co-culture (not treated with test substance) and treated co-cultures (+test substance) according to your experimental design.
Include also a culture of resting and activated TEFF alone (for example 0.2x106per well), as control for the subsequent ELISA test.
Put the plate in a 37 °C incubator for 48 hours.
At the end of cell culture, collect the supernatant in eppendorf tubes (1.5mL) and centrifuge them at 1200 x g, Room temperature, 00:05:00 in order to eliminate any residual cells.
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Collect supernatants and store them at -80°C until ELISA assay is performed.