Ifn γ elispot

Measures of antigen-specific immune response were performed by IFN-γ ELISpot (Bio-Techne, R&D Systems, Minneapolis, Minnesota, USA). Briefly, CD8+T cells were harvested from tumors of treated mice using Easy EasySep Mouse CD8+T Cell Isolation Kit (STEMCELL Technologies, Cat# 19853) following ADT treatment and added together with splenocytes from naïve FVB mice as feeder cells in a 1:10 ratio. AR25 (SRMLYFAPDLVFNEY), a 15-mer AR-specific dominant CD8+peptide epitope in FVB mice, or a pool of 62 15-mer amino acids spanning the amino acid sequence of the AR ligand-binding domain, was used as a measure for antigen-specific response measured after 48 hours of antigen stimulation. Positive control included stimulating the CD8+T cells with αCD3/αCD28 coated beads (Thermo Fisher, Cat# 11456D) at a ratio of two beads per CD8+T cell. Experimental results are shown as the number of IFN-γ spot-forming units per 10⁵ CD8+T cells normalized against media-alone control wells.

The IFN‐γ ELISpot (R&D Systems) was carrried out according to the manufacturer's protocol. Briefly, 2*10⁵ PBMC stimulated with 2*10⁴ mDC loaded with specific antigen overnight. The next day, IFN‐γ was captured and detected and the number of the positive colony in each experimental condition was counted automatically by ImmunoSpot analyzer (Cellular Technology Limited, Cleveland, OH).