For cytoplasmic extracts, the lysates were centrifuged at 12,000

For cytoplasmic extracts, the lysates were centrifuged at 12,000 g for 15 min, at 4 C. Protein concentration was measured using the Bradford assay and approximately 40 g from each sample were boiled in Laemmli buffer. Proteins were analysed on 11% SDS PAGE followed by transfer onto nitrocellulose membrane. Active caspase 3 was following website detected using a com mercially available antiserum and labelled with a HRP conjugated secondary antibody. For loading control, a monoclonal anti a tubulin antiserum was used to identify cel lular tubulin, together with an anti rat HRP conjugate. The blots were developed using Amersham ECL Kit. Confocal microscopy Control cells or cells exposed to 1, 3 or 5 puffs GPS and harvested at 1, 4 or 24 hours post exposure were fixed in 4% paraformaldehyde PBS, pH 6. 9.

The cells were perme abilised with 0. 1% Triton X 100 Inhibitors,Modulators,Libraries and non specific sites were blocked with 1% BSA in PBS. As CCRF CEM were grown in suspension, prior to staining, cells were attached onto microscope slides using Shandon Cytospin Cytocen trifuge. Active caspase 3 staining was performed using a commer cial antiserum and anti rabbit FITC antibody. Cytochrome C was identified using a monoclonal anti body and anti mouse Alexa 488 conjugate. Where necessary, nuclear counterstaining with 1 g ml propidium iodide was included. Apoptosis was induced using 2 M staurosporine. Secondary antibody negative controls were also included. Inhibitors,Modulators,Libraries The samples were visualised using a Nikon C1 Digital Eclipse Inhibitors,Modulators,Libraries Confocal Microscope system, equipped with a 488 nm Argon and a 543 Helium Neon laser through an oil immersion 60 1.

4 objective. Inhibitors,Modulators,Libraries Detection of DNA fragmentation by flow cytometry DNA fragmentation was assessed in smoke treated cells and compared to healthy cells, as well as staurosporine treated apoptotic cells using the Apo BrdU Kit. Approximately 2 106 cells were collected and briefly fixed in 1% paraformaldehyde PBS, pH Inhibitors,Modulators,Libraries 6. 9, followed by overnight fixation in 70% ethanol at 20 C. TdT catalysed end labelling of fragmented DNA with bromolated deox yuridine triphosphates was carried out at 37 C. End labelled DNA was probed with anti BrdU monoclonal antiserum provided in the kit. All cells were counterstained with a 5 g ml PI 200 g ml RNAse A solution. All samples were analysed selleck chemical Enzalutamide using a FACSCalibur cytometer and the appropriate green red filter settings. Ten thousand events from each sample were analysed. Statistical analysis All data presentations and corresponding statistical analysis was performed using SigmaPlot and SigmaStat software packages. All data in graphs are expressed as mean values SD. For one way ANOVA analysis a P 0. 05 was considered significant. Results The results described are representative of three or more independent experiments.

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