RosetteSep™ Human T Cell Enrichment Cocktail

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	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.

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.

Publication protocol

"Primary human cells were obtained from healthy adult donors at Seattle Children’s Research Institute (SCRI) and the Fred Hutchinson Cancer Research Center (FHCRC) using protocols approved by the respective Institutional Review Boards. Primary human cells used in Figures 3 and 4 were purchased from Key Biologics. 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. Whole PBMCs were used to generate cells used in Figures 3 and 4. For tests of plasma cell killing by BCMACAR T cells in vitro, autologous B and T cells were purified using EasySep Human Pan-B or T Cell Enrichment Kits (StemCell Technologies) from PBMCs obtained from either: CD34-depleted apheresis product from non-mobilized donors by the FHCRC Hematopoietic Cell Processing and Repository Core or from PBMCs collected at SCRI as described.18

Standard primary human T cell culturing conditions were incubation in a humidified environment at 37°C with 5% CO2, maintaining a density of ∼1 × 106 cells/mL by replenishing media every 2–3 days. T cell media was: RPMI-1640 with 20% fetal calf serum (Omega Scientific), 1 × GlutaMAX, 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 55 μm 2-mercaptoethanol (2ME; GIBCO), and recombinant human interleukin-2 (IL-2; 50 ng/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL) from PeproTech as described.18 Nalm-6, K562, and RPMI8226 cells (ATCC) and Nalm6-GFP (Nalm-6 transduced with LV MND GFP; Sather et al.18) were maintained in T cell media without cytokines; T cell cytokines were added for mixed cultures (below). Culturing of primary B cells for assays using in vitro generated plasma B cells is described below."

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