TITLE: PBMC- 04 - In vitro Culture of TEFF+TREG - Proliferation of TEFF
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">List of published work using this procedure:</div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. Isolate TEFF and TREG with Miltenyi Kit according to the protocol PBMC- 03.
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?. 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.
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?. Stain TEFF with CPD according to the appropriate protocol. 
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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.
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These plates can contain a volume of maximum 250µL
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?. Centrifuge TEFF and TREG at 
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Centrifuge: 1200 34, 5 min
?. Resuspend TEFF (CPD stained and unstained) and TREG in SOLUTION- 13 at a concentration of 1x106/mL.
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?. 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).
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?. 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.
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?. 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.
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?. Put the plate in a 37°C incubator for 120 hours.
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?. At the end of cell culture, collect the cells in BD tubes and centrifuge them at 
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Centrifuge: 1200 34, 5 min
?. Proceed with the FACS protocol for TEFF+TREG proliferation.