Isolation and Activation of Mouse T Cells for Chemokine Analysis

Mouse spleens were homogenized into single-cell suspensions by digesting in PBS containing 0.1% BSA and 0.6% Na-citrate, washed, and incubated with 120 Kunitz units of DNase I for 15 min following red blood cell lysis (eBioscience, 00433357)^{12 (link)}. Cells were then filtered through a 30-µm cell strainer to generate a single-cell suspension. CD4⁺ and CD8⁺ T cells were isolated using CD8a (Miltenyi Biotec, 130-104-075) and CD4 (Miltenyi Biotec, 130-117-043) T cell isolation kits, respectively. T cells were maintained at 2.5 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% FBS and 1% P/S. T cells were activated using 1.25 µg/ml anti-mouse CD3 (Fisher Scientific, 16-0031-85) and 2 µg/ml anti-mouse CD28 (Fisher Scientific, 16-0281-82) antibody treatment for 2d. Recombinant mouse midkine (R&D Systems, 9760-MD-050) was added, and Ccl4 levels were quantitated by ELISA (R&D Systems, MMB00). Conditioned medium was collected from activated T cells for chemokine assays and microglia co-culture experiments. Cell viability was measured using a commercial WST-1 cell viability assay (Millipore).