Single-cell spleen suspensions were harvested from naive C57BL/6 mice by physical disruption of 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 × 105 cells per well in phenol-free IMDM Glutamax medium supplemented with 10% FBS, 50 mM 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 mg/ml). For each well of the plates, 10 μM of compounds 2 to 5 was added, and the plates were incubated at 37°C for 6 hours. Cells that adhered to the wells were scraped first, and then Fc-block was added. Following this, the plates were put in an incubator for 30 min at 4°C. The cells were centrifuged and resuspended in a buffer containing CD11c, F4/80, CD40, CD80, and MHC-II antibodies for 30 min at 4°C. After the incubation, the plates were centrifuged and washed again. The cells were then resuspended in 0.5 ml of fluorescence-activated cell sorting (FACS) buffer (PBS, 0.02% sodium azide, 0.5% bovine serum albumin). The mean fluorescence intensity for CD11C or F4/80 cells and activation markers CD40 and MHC-II was used to identify the maturation of DCs or macrophages.

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