APC maturation evaluation

Antigen-presenting cells maturation study 

Single-cell spleen suspensions were harvested from Swiss naive mice by physically disrupted their spleens and passing the disrupted spleens through stainless-steel mesh. Red blood cell lysis buffer was used to lyse the erythrocytes. Then, a 96-well plate was filled with 2 x10⁵ cells/well in phenol-free IMDM Glutamax medium supplemented with 10% fetal bovine serum, 50 mM 2-mercaptoethanol, 100 U/mL penicillin and 100 mg/mL streptomycin. For each well of the plates, 10 μM of vaccine candidates was added, and the plates were incubated at 37 °C for six hours. Cells that adhered to the wells were scraped first, and then Fc-block (eBioscience, California, United States) was added. Following this, the plates were put in an incubator for 30 minutes at 4 °C. The cells were centrifuged and resuspended in a buffer containing CD11c, F4/80, CD40, CD80 and MHC-II antibodies for 30 minutes at 4 °C. After the incubation, the plates were centrifuged and washed again. The cells were then resuspended in 0.5 mL of FACS buffer (PBS, 0.02% sodium azide, 0.5% BSA) using an LSR II flow cytometry (LSR II Flow cytometer, BD Biosciences, California, United States). Both percentage fluorescence positive and mean fluorescence ontensity for CD11C or F4/80 cells and activation markers CD40, CD80 and MHC-II were used to identify the maturation of dendritic cells or macrophages.