Biomag goat anti rat igg magnetic beads

PbT-II and gDT-II CD4⁺ T cells were negatively enriched from the spleens and lymph nodes of PbT-II/uGFP or gDT-II mice as described (Smith et al., 2003 (link)). Briefly, tissues were disrupted by passage through 70 ​μm cell strainers and red cells were lysed. Single cell suspensions were labelled with a cocktail of rat monoclonal antibodies (WEHI Antibody Services, Melbourne, Australia) specific for CD8 (clone 53-6.7), MHC Class II (clone M5/114), macrophages and dendritic cells (clones BM8 [anti-F4/80] and M1/70 [anti-CD11b]) granulocytes (clone RB6-8C5 [anti-Gr1]) and red blood cells (clone Ter119 [anti-Ter-119]) prior to incubation with BioMag goat anti-rat IgG magnetic beads (Qiagen, Hilden, Germany) and separation of labelled, non-CD4 T cells using a magnet. Enriched naïve PbT-II CD4⁺ T cells were counted and their purity was analysed by staining with anti-CD4, anti-Vα2 and anti-Vβ12 TCR antibodies. Negatively enriched gDT-II/uGFP cells were then positively enriched using the Invitrogen Dynabeads/DETACHaBEAD Mouse CD4 Kit (Life Technologie AS, Oslo, Norway) following the manufacturer's instructions. Cell counts were adjusted to 2.5 ​× ​10⁵/mL in PBS and 0.2 ​mL were injected i.v. into recipient mice. In some instances, cells were labelled with CellTrace Violet (Thermo Fisher) prior to transfer following the manufacturer's instructions.