EasySep™ Human T Cell Isolation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
cells were counted, centrifuged at 300 g for 5 min at 4°C.	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.	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.

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.

Publication protocol

STEMCELL Technologies EasySep Human T Cell Isolation Kit (Cat #17951). Protocol was followed as per manufacturer’s instructions. Briefly, cells were counted, centrifuged at 300 g for 5 min at 4°C. 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. 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. Isolated T cells were collected by pouring off in one quick motion and centrifuged at 300 g for 5 min at 4°C. Pellet was resuspended in 1 ml of 1X PBS buffer for counting and flow cytometry.

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