1% CJ-023423 cost bovine serum albumin in 1 PBS, the cells were incubated with rabbit KIF20A polyclonal antibody or rabbit -tubulin polyclonal antibody at 4C overnight. Subsequently, the cells were washed three times with 1 PBS and stained with DyLight-labeled goat anti-rabbit IgG at room temperature for 4 hours. Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride; Vector Labs, Burlingame, CA, USA). The cells were then examined and photographed using a laser scanning confocal microscope. The excitation wavelength was 488 nm for DyLight and 405 nm for DAPI. Images were merged using ZEN software. were gently washed with HEPES-buffered saline and then incubated in Fluo 4-AM working solution for 30 minutes at 37C in a cell incubator. Next, the cells were washed with HEPES-buffered saline to remove extracellular Fluo 4-AM and incubated for an additional 20 minutes to allow deesterification of Fluo 4-AM into its active-dye form Fluo 4. Analysis was performed on a BD FACSCaliburTM system at an excitation of 488 nm. For intracellular sodium and potassium ion measurements, 2 L of 2.5 mM SBFI-AM and PBFI-AM stock solutions were individually added to 1 mL of cells at a final concentration of 5 M 1 hour prior to conducting the experiment. Incubation was continued at 37C in a 5% CO2 atmosphere. Immediately before FCA, propidium iodide was added at a final concentration of 10 g/mL. Approximately 1 104 cells were analyzed by sequential excitation of the cells at 355 nm for SBFI-AM or PBFI-AM and 488 nm for PI, using a BDTM LSR II Flow Cytometer System. Results and discussion 
Detecting apoptosis with Annexin V and JC-1 assays Apoptotic cells display significant externalization of PS, which can be validated using the Annexin V assay. As shown in Supplementary Measurements of intracellular calcium, sodium, and potassium using flow cytometry Intracellular calcium ion concentration was measured using Fluo4-AM and 20% pluronic F-127 dissolved in DMSO to make a 1 mM stock solution. The decreasing trend of membrane capacitance observed in our experiments is consistent with the observations of previous studies.23,24 Similarly, we calculated that cytoplasmic conductivity decreased from 0.217 0.005 to 0.120 0.006 S/m over the 12-hour time course based on the equation,25 where s indicates the relative permittivity of the surrounding solution is a constant, about 81. This generally agrees with previous studies examining Jurkat T-cells undergoing etoposide-induced apoptosis.23 Compared to the Annexin V and JC-1 assays, the DEP assay can be used to monitor apoptosis noninvasively without the use of added markers or labels. Gene expression profiling during Ara-C-induced apoptosis To better understand the molecular mechanism underlying apoptosis using DEP monitoring, we examined mRNA changes in NB4 cells after Ara-C treatment over a 12-hour time course using a human whole genome oligo array. Furthermore, we analyzed expression changes in terms of transcriptional regulation of defined gene networks and pathways and present a model of the dynamic and temporal network of pathway activities related to Ara-C-induced apoptosis, which may have implications for identifying potential new effectors useful for treating APL. Here, we PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19818408 used DEP analysis to systematically investigate potential mechanisms responsible at the gene level and at the cellular level for changes in membrane capacitance and cytoplasmic conductivity occurring during apoptosis. As described above, memb
Posted inUncategorized