Cell Isolation Human T cells

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Upstream tips
cells were counted, centrifuged at 300 g for 5 min at 4°C.
Protocol tips
Pellet was resuspended in EasySep buffer (Catalog #20144) and incubated with DNase I solution (Catalog #07900) at a concentration of 100 μg/ml at room temperature for 15 min prior to labelling and separation. Following incubation, any cell aggregates were filtered out using 37 μm cell strainer (Catalog #27250). Cells were then diluted to a cencentration of 5 × 107 cells/ml in EasySep buffer. Sample was transferred to 5 ml polystyrene round-bottom tube (Catalog #38007) and isolation cocktail was added at a concentration of 50 μl/ml.
Downstream tips
Sample was mixed and incubated at room temperature for 5 min. RAPIDSPHERES were thoroughly vortexed and added to the sample at a concentration of 40 μl/ml. Following this, the cell solution was topped up to 2.5 ml with EasySep buffer, mixed and placed on the EasySep magnet (Catalog #18000) for 3 min at room temperature.
Protocol tips
Flow cytometric analysis of isolated T cell sample purity obtained using the Pan T cell isolation kit.
Downstream tips
Isolated T cells were further activated by 1-week culturing in RPMI 1640 supplemented with 10% FBS along with Dynabeads Human T-Activator CD3/CD28 (Veritas, DB11131) and IL-2 (Corning, 354,043) in a 37 °C, 5% CO2 atmosphere.
Protocol tips
Following the manufacturer's instructions
Protocol tips
Briefly, PBMCs were selectively depleted of CD16, CD19, CD20 cells and were discarded (Dynabeads® Untouched™ Human T Cells Kit, Invitrogen, Carlsbad, CA, USA). The purity of T cell enrichment was checked using flow cytometry
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.
Protocol tips
For most of the experiments presented here, T cells were enriched directly from whole peripheral blood using a negative selection kit (RosetteSep Human T Cell Enrichment Cocktail, STEMCELL Technologies), either freshly prepared or thawed from stocks frozen at 2–4 × 107 cells/mL in culture media with 10% DMSO.
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