Pan t cell isolation kit

Mature CD3⁺ T-cells were isolated from the spleens of male or female WT mice using Pan T-cell isolation kits (Miltenyi) and negative magnetic sorting for CD3⁺ isolation. The CD4⁺ and CD8⁺ T-cell subpopulations were then isolated by flow cytometry, as described previously [28 ].

All biotinylated and fluorescent antibodies, human CD138 microbeads, and Pan T Cell Isolation Kits were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). DMEM, RPMI-1640, L-glutamine, penicillin-streptomycin, and phosphate buffered saline (PBS) were purchased from Corning (Corning, NY). Fetal bovine serum, live-cell dyes, lipophilic tracers, collagenase, and counting beads were purchased from Life Technologies (Carlsbad, CA). 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG2000), and polycarbonate membranes were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and chloroform were purchased from Millipore Sigma (Burlington, MA). Streptavidin conjugation kit was purchased from Abcam (Cambridge, United Kingdom). Human Cytokine Array Q1 was purchased from Raybiotech (Peachtree Corners, GA). All mice used in this study were NCG (strain: 572), female, 50–56 days old, and purchased from Charles River (Wilmington, MA). All mice experiments in this study was in compliance with the Institutional Animal Care and Use Committee at Washington University.

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.

Human PBMCs and murine splenocytes were cultured in complete RPMI with 10% FBS, 1% Pen-Strep and stimulated by addition of anti-CD3 with or without anti-CD28 to cell cultures (eBioscience). For selected experiments, murine splenocytes were cultured with 0.03ug/mL IFNγ (eBioscience). In some cases, T cells were purified using pan T cell isolation kits (Miltenyi Biotec) and stimulated with plate-bound anti-CD3 and anti-CD28 (eBioscience) or anti-CD3, anti-CD2 and anti-CD28 coated beads (Miltenyi Biotec).