Phenotypic Modulation of Dendritic Cells by Botanical Compound

To assess the effects of BC on immune cells, D1DCs (a murine dendritic cell line) [22 (link)] were cultured with increasing amounts of BC extracts (up to 20 mg/mL). After 48 h, the D1DCs were detached using PBS/EDTA, washed with FACS buffer (PBS with 0.5% BSA and 0.02% sodium azide) and stained at 4 °C for 30 min with the following antibodies: anti-CD40-APC (Biolegend, San Diego, CA, USA, clone 3/23, #12-4612), anti-CD86-FITC (eBioscience, San Diego, CA, USA) clone GL1, #11-0862-85,) and anti-MHC II-PE (eBioscience, San Diego, CA, USA) clone M5/114.15.2, #12-5321-82). 7-AAD (Invitrogen, Waltham, MA, USA, #A1310) was included to stain the dead cells. Thereafter, the cells were washed with FACS buffer to remove unbound antibodies and resuspended in 100 μL FACS buffer. FACS analysis was performed on an LSR-II cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed with FlowJo V10.