Dynal cd4 t cell negative isolation kit

CD4⁺ T cells were purified from spleens by magnetic‐activated cell sorting (Dynal CD4⁺ T cell negative isolation kit, Invitrogen, Gent, Belgium) according to the manufacturer's protocol. Naive or memory T cells were purified from previously isolated CD4⁺ T cells subsets by positive or negative selection of CD62L‐expressing cells using the magnetic cell sorting kit (CD62L microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. For Treg differentiation experiments, CD25‐positive cells were removed from purified CD4⁺ T cells by magnetic‐activated cell sorting using FITC‐conjugated anti‐CD25 antibodies and anti‐FITC microbeads (Miltenyi Biotec).

CD4⁺ T cells were purified from spleen by magnetic-activated cell sorting (Dynal CD4⁺ T cell negative isolation kit; Invitrogen). For the two-dimensional gel electrophoresis, spleen cells were incubated with FITC-conjugated anti-mouse TCRVβ6 antibody (RR4-7; BD Pharmingen), then with anti-FITC magnetic beads (Miltenyi Biotec) and sorted by magnetic sorting according to the manufacturer’s protocol. T cell purity assessed by flow cytometry was >92%.