Bromodeoxyuridine brdu incorporation assay

To determine the anti-proliferative effect of WA, the cancer cells were treated in various concentrations of WA and/or dimethyl sulphoxide (DMSO) for 24 h followed by MTT ((3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay as per the manufacturer's protocol (Sigma Aldrich, Saint Louis, MO, USA). Cell proliferation was quantified for the selected clones stably expressing pCMV/HCT-116 or AKT/HCT-116 using the Bromodeoxyuridine (BrdU) incorporation assay (Cell Signaling, Danvers, MA, USA), according to the manufacturer's protocol, and the trypan blue exclusion method [51 (link)]. Anchorage-independent growth was monitored by colony formation assay using a CytoSelect^™ 96-well in vitro tumor sensitivity assay kit (Cell Biolabs, Inc, San Diego CA, USA). The cell harvesting and assay were performed as per the manufacturer's instructions using 5 × 10³ cells (pCMV/HCT-116 and AKT/HCT-116) [24 (link)].

For the mixed rat peripheral blood lymphocyte reaction, 1 × 10⁵ peripheral blood lymphocytes (rPBLs) isolated from healthy rats were added into 100 μl α-MEM and plated on each well in 96-well plates. After 4 h of culture, different doses (0, 50 μg/μl, 100 μg/μl, and 200 μg/μl) of hFMSC secretome and hAMSC secetome in 100 μl α-MEM were added into each well. rPBSL culture with serum-free α-MEM served as the baseline control. After an additional 1, 3, and 5 days of culture, the proliferation of rPBLs was determined by bromodeoxyuridine (BrdU) incorporation assay according to the manufacturer’s manual (Cell Signaling Technology, USA). Optical density was measured at 450 nm.