Accumax dissociation solution

Since the main constituents of TE and RE (curcumin and carnosic acid, respectively) have been implicated as antioxidants, Dihydrorhodamine123 (DHR123; Invitrogen, Carlsbad, CA, USA) assay was used to determine the amount of reactive oxygen species (ROS) present after 12 h treatment with each extract according to literature [19 (link)]. Briefly, cells were detached using Accumax dissociation solution (Innovative Cell Technologies), collected and centrifuged for 10 m at 500 rcf at 4 °C. The pellet was washed once with PBS before resuspension in 1 mL of stain (30 μM DHR123 in DMEM). The cell suspension was then incubated at 37 °C for 30 m, pelleted, and resuspended in 1 mL DMEM and filtered before fluorescence analysis of cells.

Rapidly cycling eGFP AR reporter cells were collected using Accumax dissociation solution (Innovative Cell Technologies, San Diego, CA), and dead cells were counterstained with DAPI (Invitrogen, Grand Island, NY). For FACS-sorting of ARsig-lo and ARsig-hi cells, 5% of the entire population with lowest and highest eGFP expression was sorted out using BD FACSAria cell sorter. The 5% cutoff was used because it generates at least a 100-fold difference in median AR-GFP reporter signal between ARsig-lo and ARsig-hi cells and also allows us to have sufficient numbers of sorted cells to conduct various assays. For flow cytometric analysis of reporter activity, eGFP expression was measured using the BD-LDRII flow cytometer and analysis was done using FlowJo software.

Vero cells (JCRB9013) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Note that the quality of the cells was assured by standard tests of the cell bank. Cells were cultivated in a normal growth medium consisting of Eagle’s minimal essential medium (MEM; Fujifilm Wako Pure Chemical Co., Osaka, Japan) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, MO, USA) and penicillin–streptomycin (Fujifilm Wako Pure Chemical Co.), at 37 °C and under an atmosphere of 5% CO₂ and 100% humidity. Cells were detached with AccuMax dissociation solution (Innovative Cell Technologies, CA, USA) and passaged at a split ratio of 1:5.