Ter the addition of trypan blue were Zellvitalit Th by Z Select a minimum of 300 total cells determined per data point. The data were to be determined using PHA-739358 Aurora Kinase inhibitor GraphPad Prism software IC50 values. The method of Chou and Talalay was used to assess the synergies and the indices were calculated using a combination CalcuSyn V2 software. CI values were less than 1.0 as evidence for synergy. In some experiments, Zelllebensf Ability by flow cytometry Annexin VF Intended coloring. For these experiments were 22A or 22B Unified Messaging Unified Messaging cells in six-well plates T seeded and treated for 48 h with ABT 737, cisplatin, etoposide, or combinations of ABT 737 and chemotherapy. After the treatment were adh Rente cells from the plates using trypsin detached St and, together with floating cells.
The cells were then washed twice with cold PBS JNJ-38877605 943540-75-8 and resuspended in 1 annexin V binding buffer at a concentration of 1106 cells per ml Single cell suspensions were then transferred to 5 ml of culture and found Rbt with 5 L of annexin V-FITC and 5 l propidium iodide for 15 min at room temperature in the dark. After the dyeing F Was an annexin V binding buffer in each R Hrchen added and the samples were placed on ice. Two-color flow cytometry analysis were performed with a Coulter Epics XL flow cytometer. Clonogenic assays. The cells were seeded in bo t Your 100 mm, tightened overnight and then treated for 1 h with ABT 737 or cisplatin alone or in combination. After treatment, the cells were washed twice with PBS and separated from the plates using trypsin.
Single cell suspensions were diluted plated in DMEM with 10% FBS and an equal number of cells in six-well plates. The cells were cultured for 12 to 15 days, then for 30 min colonies in an L Solution of 6% glutaraldehyde and 0.5% crystal violet Fnd Rbt were dissolved in water. The plates were washed with water until no more dye was detected in the liquid rinse Age. After drying in air, colonies consisting of 50 or more cells hlt gez. 1232, Li et al. Immunoblotting. For immunoblotting experiments, the treated cells scraped off the plates, centrifuged at 1300 rpm for 5 min at 4, washed once with cold PBS, then lysed for 10 min on ice in 150 mM NaCl, 50 mM Tris pH 8.0, 0 , 1% SDS, 1 40% Nonidet P, 20 g / ml aprotinin, 3 g / ml leupeptin and 1.5 mM phenylmethylsulfonyl fluoride.
The lysates were at 14,000 rpm for 2 min and centrifuged, and the four whichever type Walls were transferred to new R Hrchen. Bio-Rad protein assay dye concentrate was then used to determine protein levels in the lysates. For detection of the cleavage products of caspase 3 and determining the protein content of the Bcl-2, Bcl XL, Mcl 1, Bax, Bak, Noxa, Bik and actin proteins were A subject electrophoresis gels 12.5% SDS-PAGE. For detection of the cleavage products of PARP and Mcl 1L levels, the proteins were Electrophoresed on 10% SDS-PAGE gels subjected. After electrophoresis, the proteins were To nitrocellulose membranes for 3 h at 45 V and 4 transmitted. The membranes were blocked at room temperature for 1 h in TBST buffer containing 5% skim milk.
The membranes were blocked with TBST buffer probed overnight at 4 with rpern prime Ren Antique, Washed again in TBST, then probed for 1 h at room temperature with secondary Rantik Washed body. After the conclusive The four W Rule in TBST, the membranes were developed with verst Rkter chemiluminescence reagent, as instructed by the manufacturer. When blots were probed with actin to mpfen Ampicillin, they were stripped by incubation for 45 min at 37 in 0.1 M glycine, pH 2.9. The stripped