Dynabeads untouched human t cells

All animal procedures were approved by the Institutional Animal Care and Use Committee at UC San Diego. Primary murine T cells were freshly isolated from C57BL/6J mice. A single cell suspension of splenocytes was created and T cells were isolated via magnetic depletion using CD4⁺ or pan-T cell isolation kits (Miltenyi Biotec) according to the manufacturer’s recommendations. Murine T cells were not cryopreserved as the cells were found to be sensitive to DMSO at concentrations greater than 1 %(v/v) and reconstituted murine T cells expanded less than freshly isolated cells (data not shown).
Primary hT cells were isolated from anonymous donor blood concentrate enriched in the buffy coat (Stanford Blood Bank and San Diego Blood Bank). PBMCs were enriched from buffy coat within 24 hours of collection using density separation in a Ficoll gradient (Lymphopure). PBMCs were then transferred to serum-free cell freezing media (Bambanker) and frozen overnight at −80 °C. Aliquots were then stored in liquid nitrogen and used within 6 months. Pan T cells were isolated from thawed PBMC aliquots using magnetic depletion (Dynabeads Untouched Human T cells, Invitrogen) according to the manufacturer’s recommendations.

T cells from peripheral blood of healthy human donors (Fig. 4k) were prepared as described previously^{14 (link)}. Briefly, peripheral blood mononuclear cells (PBMCs) from anonymous, healthy donors (New York Blood Center) were isolated by Ficoll gradient separation. CD14⁺ monocytes were removed from the PBMC fraction by positive selection. The remaining negative fraction was used to isolate T cells.
After PBMCs from 20 healthy donors were mixed prior to sorting T cells, naïve T cells were isolated with Dynabeads^™ Untouched^™ Human T cells (Invitrogen) according to the manufacturer’s protocol. Sorted human T cells were frozen with FBS containing 10% dimethyl sulfoxide (Fisher Scientific) in liquid nitrogen tank until the day of FACS sorting.