Anti mouse cd4 clone l3t4

Antigen-specific CD4⁺ T cells and CD8⁺ T were isolated from OT-II and OT-I splenocytes respectively, through magnetic bead separation. Anti-mouse CD4 (clone L3T4) and CD8 (clone Ly-2) microbeads were obtained from Miltenyi Biotec Ltd (Bergisch Gladbach, Germany), and used according to the manufacturer's instructions. The MHC class I peptide OVA_257–264 (SIINFEKL) (JPT Peptide, Berlin, Germany) and the MHC class II peptide OVA_323–339 (ISQAVHAAHAEINEAGR) (Mimotopes Pty Ltd, Clayton, VIC, Australia) of ovalbumin (OVA), were used as the target antigens for CD8⁺ CTL and CD4⁺ Th cells, respectively. Unless otherwise stated, 1 μg l⁻¹ peptide concentration was used for both CD4⁺ and CD8⁺ cell stimulation. Non-adherent and loosely adherent cells were harvested, washed, and resuspended at 1 × 10⁶ cells per ml in DC medium; OVA_257-264 or OVA_323-339 were added to the cells and incubated for 4 h at 37 °C/5% CO_2. Free peptides were washed off with dulbecco's phosphate-buffered saline (DPBS), and the DCs were resuspended in cIMDM-5 at 1 × 10⁶ cells per ml for T-cell stimulation.

Antigen-specific CD4⁺ and CD8⁺ cells were isolated from OT-II and OT-I splenocytes respectively, through magnetic bead separation. Anti-mouse CD4 (clone L3T4) and CD8 (clone Ly-2) microbeads were obtained from Miltenyi Biotec Ltd (Bergisch Gladbach, Germany) and used according to the manufacturer’s instructions. The MHC class I peptide OVA_257–264 (SIINFEKL) (JPT Peptide, Berlin, Germany) and the MHC class II peptide OVA_323–339 (ISQAVHAAHAEINEAGR) (Mimotopes Pty Ltd, Notting Hill, VIC, Australia) of OVA were used as the target antigens for CD8⁺ CTL and CD4⁺ Th cells, respectively.