In Vitro CD4+ T Cell Proliferation Assay

The CFSE Cell Division Tracker Kit (Biolegend) was used for flow cytometry analysis of in vitro CD4⁺ T cells proliferation assay according to manufacturers' protocol. Briefly, autologous lymphocytes were pre-treated with 0.1 μM CFSE before being added to MDDC cultures in the presence of the lymphocyte mitogen Concanavalin A (5 μg/mL; Sigma Aldrich, Merck) for 120 h (32 (link)). At the end of assay, cells were then labeled for anti-CD3 APC (MEM-57) and anti-CD4 PE (RPAT4) (BD Biosciences). The Live/Dead Fixable Cell Stain Kit was added to the assay according to the manufacturer's instructions. Cells were then washed twice with PBS and resuspended in 200 μL of 4% Formaldehyde-PBS to proceed to flow cytometry analysis as above-mentioned.