Anti 6 4pp clone 64m 2

The assay of NER capacity was modified to a cell-based fluorescent assay that evaluates direct binding of an antibody specific for UV-induced 6-4PP (29 (link), 30 (link)). Briefly, cells were seeded in a 24-well plate at a density of 100,000 cells per well. Cells were incubated for 4 h with drugs. Cells were irradiated with UV-C (80 J/m², Stratalinker 1800, Energy mode). Following UV irradiation, cells were incubated with culture medium and collected at 0 h, 2 h, 4 h and 8 h. Immunofluorescent staining (IF) was performed as described in the product protocol (Anti-6-4PP Clone 64M-2 1:500, COSMO BIO). Immunofluorescent images were captured and processed using CellProfiler (31 (link)). Mean fluorescence intensity of the level of 6-4PP at 2 h, 4 h or 8 h were normalized to 100% intensity of 6-4PP at 0 h (11 (link)). NER capacity at 2 h, 4 h and 8 h were represented as % of 6-4PP removal (100% minus 6-4PP% at 2 h, 4 h and 8 h after UV). For measuring NER in 3D PDOs, organoids were disassociated, and cells were plated overnight as 2D adherent cell culture before 6-4PP assay. NER capacity in PDOs was measured by double immunofluorescence (IF) staining with rabbit anti-cytokeratin 7 (CK7) and mouse anti-6-4PP. Only CK7 positive cells (urothelial cells) were counted for NER capacity (32 (link)).