mESCs were processed for the determination of cell viability after various times of NaF exposure using the Cell Counting Kit 8. In this assay, water soluble tetrazolium 8 is produced by living cells and thus the level of WST produced is proportional to the viability of cells. All experimental methods were adopted in line with the manufacturers guidelines and WST absorbance was CX-4945 Protein kinase PKC inhibitor measured at 450 nm using a microplate reader. The degree of DNA synthesis in mESCs was measured by the addition of 1 uCi of 3H thymidine deoxyribose to the cells cultured in 96 well plates over the last 4 h before cell harvesting. Cells were obtained utilizing a harvester 24 h after NaF publicity. Beta emission from the 3H TdR involved cells was tested for 1 minute using a liquid scintillation counter. JNK activity was determined utilizing an immunometric assay kit according to the manufacturers instructions. In quick, mESCs were stopped in a cell lysis buffer. Protein concentrations were determined utilizing a BCA protein assay kit and samples containing equal levels of protein were placed in to p SAPK/JNK sandwich Gene expression ELISA kit microtiter plates. Finally, the absorbance was measured using a microplate reader. Cell cycle was based on flow cytometric analysis after propidium iodide staining. In brief, NaF treated cells were fixed with 70-300 ethanol for 24 h, and then incubated over night at 4 C with 500 ul of the PI staining mixture. After discoloration, 10,000 cells per experiment were examined using the FACS Calibur system. Cell cycle progression was determined utilizing the ModFit LT plan. The mESCs were washed twice with phosphate buffered saline before suspension conjugating enzyme in 1 binding buffer. FITClabeled annexin V was mixed with 100 ul of the mobile suspension containing 2 105 cells, and the cells were incubated at room temperature for 5 min. Afterwards, 4 ul of PI solution was added to the cells accompanied by an additional 5 min incubation. The spread details of the cells were analyzed using a FACS Calibur program. Four cell populations were identified based on the following characteristics, the viable population in the lower left quadrant, the early apoptotic population in the lower right quadrant, the necrotic population in the upper left quadrant, and the late apoptotic or necrotic population in the upper right quadrant. DNA fragmentation in NaF uncovered mESCs was assayed using a Cell Death Detection ELISA package and all processes were done based on the manufacturers directions. The mESCs were then stained with 50 nM and washed twice with 1 ml PBS 3,3 dihexyloxacarbocyanine iodide for 20 min at 37 C. Fluorescence linked to MMP was measured using a FACS Calibur process, and the change in MMP level was determined using the Window Multiple Document Interface 2. 9 Software. A stock solution of 2,7 dichlorodihydrofluorescein diacetate was prepared in DMSO and stored at 20 C in the dark. The mESCs exposed to NaF were incubated with 25 uM DCFH DA for 20 min.