MagniSort™ Human T cell Enrichment Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	According to the manufacturer’s instructions.	After T-cell enrichment, 0.5 × 106 T cells were added to 1 mL of Roswell Park Memorial Institute (RPMI) culture medium with HEPES and L-Glutamine (Lonza, Basel, Switzerland) supplemented with gentamycin (Lonza), in round-bottomed polypropylene tubes.

Protocol tips
According to the manufacturer’s instructions.
Downstream tips
After T-cell enrichment, 0.5 × 106 T cells were added to 1 mL of Roswell Park Memorial Institute (RPMI) culture medium with HEPES and L-Glutamine (Lonza, Basel, Switzerland) supplemented with gentamycin (Lonza), in round-bottomed polypropylene tubes.

Publication protocol

Expression kinetics were determined by measuring ICs at defined time points after T-cell stimulation ex vivo. Briefly, peripheral blood mononuclear cells were isolated from blood collected in heparin tubes by density gradient centrifugation using Lymphoprep (Alere Technologies AS, Oslo, Norway). Next, T cells were isolated by negative selection using the MagniSort Human T cell Enrichment Kit (Thermo Fisher Scientific, Waltham, MA., USA) or the EasySep™ Human T cell isolation kit (Stemcell Technologies, Vancouver, Canada) (results were found to be similar for both kits, purity ≥92%) according to manufacturer’s instructions. After T-cell enrichment, 0.5 × 106 T cells were added to 1 mL of Roswell Park Memorial Institute (RPMI) culture medium with HEPES and L-Glutamine (Lonza, Basel, Switzerland) supplemented with gentamycin (Lonza), in round-bottomed polypropylene tubes. Gibco Dynabeads™ Human T-activator anti-CD3/anti-CD28 (ThermoFisher, Waltham USA) were added to T cells in a ratio of 1:5. Stimulated T cells were then incubated at 37 °C, 5% CO2 and collected after 1,2,3,4, 18, 42, 66 and 90 h and stained for CD8, CD45RA, CD25, PD-1, VISTA, and CD40L. In parallel, unstimulated T cells were also assessed for the same ICs including CD28 at each time point (See additional Table 2 for the antibodies used). Staining was performed as described above.

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Manufacturer protocol

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