2.6. Effect of calcitriol administration on RANKL expression in Th cells in an inflammatory environment

In parallel to the investigation of cell polarization, the gene and protein levels of RANKL in the Th cells (LPS group, LPS + DC group and LPS + DC + Cal group) following a 5‐day incubation were determined by qRT‐PCR and Western blot analysis (for details, see Sections 2.8 and 2.9). In addition, immunofluorescence and flow cytometry assays were performed to assess the proportion of IL‐17⁺/RANKL⁺ cells. 13 Briefly, after Th cell smears were prepared, Th cells were incubated with primary antibodies against RANKL (ab45039; Abcam) and IL‐17 (ab180904; Abcam) overnight at 4°C and then incubated with secondary antibodies (Alexa Fluor 488‐conjugated goat anti‐rabbit and Alexa Fluor 647‐conjugated goat anti‐mouse) for 50 minutes in the dark. Finally, the cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and then visualized and imaged with a fluorescence microscope (NIKON ECLIPSE C1, Nikon DS‐U3). Similarly, for detecting the proportion of IL‐17⁺/RANKL⁺ cells by flow cytometry, cells were immunostained with PE‐conjugated anti‐mouse RANKL (IK22/5, BioLegend) and APC‐conjugated anti‐mouse IL‐17 antibodies, and the immunostained IL‐17⁺/RANKL⁺ cells were detected and analysed with a Beckman Coulter Epics XL flow cytometer (Beckman Coulter). 32