Nov 21, 2020
Version 2
PBMC- 04 - In vitro Culture of TEFF+TREG - Proliferation of 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- 04 - In vitro Culture of TEFF+TREG - Proliferation of TEFF. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxvmpn6Version 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: 44757
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
Guidelines
Work under laminar flow hood when you are processing samples, from the beginning to the end of the following procedure.
Materials
MATERIALS
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Penicillin/StreptomycinEuroCloneCatalog #ECB3001D - 100 ml
CPD-eFluor670 - 500 µgeBioscienceCatalog #65 0840 85
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). Leave TREG cells in their SOLUTION- 13 and proceed with TEFF cells.
Document
NAME
SOLUTION- 13 - Complete culture medium
CREATED BY
Farmacologia Medica
Stain TEFF with CPD according to the appropriate protocol.
Note
IMPORTANT!
It is necessary to have an initial number of TEFF of at least 1x106 for staining.
Include in your experiment alsoTEFF cells unlabeled with CPD, as fluorescent background control for FACS analysis (see the appropriate protocol in flow cytometry).
Use sterile 96-well round bottom plates.
Note
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 (CPD stained and unstained) 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 (CPD stained and unstained) with PHA 5µg/ml (final concentration) and IL-2 40 ng/mL (final concentration) by diluting the stock aliquots. Leave also wells of TEFF (CPD stained an dunstained) unstimulated (resting control).
Put TEFF-CPD labeled cells and TREG cells in the 96-well plate at a ratio of 1:1(for example, 0.1x106TEFF+0.1x106TREG) 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 stained and unstained CPD (for example 0.2x106 cells per well), as control for the subsequent flow cytometric analysis.
Put the plate in a 37°C incubator for 120 hours.
At the end of cell culture, collect the cells in BD tubes and centrifuge them at 1200 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Proceed with the FACS protocol for TEFF+TREG proliferation.