Negative selection magnetic bead isolation kit

Bone marrow-derived DC (BMDC) were generated, LPS-matured and OVA-pulsed as previously described
^{24 (link)}. Briefly, bone-marrow (BM) cells were extracted from the femur and tibia of mice. To generate BMDC, BM cells were cultured over 7 days in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Gibco) in the presence of 20 ng/ml GM-CSF (BioSource). For all experiments, BM DCs were CD11c selected using magnetic bead separation (Miltenyi Biotech). To induce DC activation, CD11c+ DCs were matured overnight with 100ng/ml LPS (Sigma). BMDC were blocked for 30 min at 37°C with anti-IL-15Rα (AF551) or isotype-matched controls (both R&D Systems, 20μg/ml unless otherwise stated). DC were nucleoporated with 5μg lentiviral plasmid DNA using the Amaxa Mouse Dendritic Cell Nucleofector Kit (Lonza). Unsorted cells were used for experiments. Splenic CD4+ T cells were isolated using a negative selection magnetic bead isolation kit (Miltenyi Biotech). For proliferation experiments, CD4+ T cells were labeled with 5μM CFSE dye for 20 min at 37°C then washed before co-culture.

BMDCs from C57BL/6 (WT), WASKO, or phosphorylation-null WASp knockin (Y293F [40 (link)]) mice were generated, matured with LPS, and pulsed with OVA, as described previously [7 (link)]. CD4⁺ T cells were isolated from spleen and lymph nodes of OT-II mice (OVA_323–339 peptide/I-A^b-specific CD4 T cells; Charles River Laboratories, Wilmington, MA, USA) by use of a negative selection magnetic bead isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). All animals were handled in strict accordance with good animal practice, as defined by UK Home Office Animal Welfare Legislation, Animals (Scientific Procedures) Act 1986, and all animal work was approved by the Institutional Research Ethics Committee (Institute of Child Health, University College London, United Kingdom) and performed under Project License Number 70/7024. For confocal microscopy, DCs and T cells were mixed in a 1:5 ratio and centrifuged gently at 30 g for 5 min to enhance contact formation. The suspension was incubated at 37°C for an indicated time; loose pellets were resuspended gently and seeded on poly-l-lysine-coated glass coverslips for staining and analysis.