Dendritic Cell (DC) Maturation Assay

Immature BMDCs (5 × 10⁵) were incubated in 6-well plates ± TPCS_2a overnight at 37°C, 5% CO₂ in the dark. TPCS_2a was washed (PBS, 3×) from the cells and cells were incubated for additional 4 h before light treatment. Lipopolysaccharide (LPS) from E. coli (100 ng/ml, Sigma) was used as a positive control to induce BMDC maturation, untreated cells were used as negative control. 18 h post light treatment, cells were scraped and stained with fluorescence-labeled anti-CD11c (FITC or PE, clone HL3, BD Biosciences), anti-MHC class II (I-Ab, Alexa Fluor 488 or APC, clone AF6-120.1, BioLegend), and anti-CD86 (PE, clone GL1, BioLegend) antibodies. Unlabeled anti-CD16/32 (clone 93, eBioscience) was used to block Fc receptor binding. Flow cytometric analysis of the cells was performed on a BD LSR II flow cytometer (BD Biosciences) and data analysis performed using FlowJo software (v10.0.7, FlowJo, LLC).