( 1. Treat 5 x 105/ml Jurkat cellswi...)Apoptosis detection by annexin V and,,,active caspase-3 with the Agilent 2100,,,bioanalyzer

1. Treat 5 x 105/ml Jurkat cells with 1 M camptothecin or 0.5 g/ml anti Fas antibody for 224 hours to induce apoptosis. 2. Harvest control and camptothecin- treated or Fas antibody- treated cells and adjust cell density to 1 x 106 cells /ml in staining buffer. 3. Add 1.5 l SYTO16 per ml and incubate 10 minutes at 37 C. 4. Wash cells in 2 ml staining buffer and centrifuge (500 x g, 5 minutes). 5. Resuspend 5 x 105 cells in 500 l 1x BD permeabilizing solution (dilute in distilled water) and incubate 10 minutes at RT. 6. Wash cells in 2 ml staining buffer and centrifuge (500 x g, 5 minutes). 7. Add anti caspase-3 antibody at 5 g/ml in a total of 100 l staining buffer to 1 x 106 cells. Incubate 20 minutes on ice. 8. Wash cells in 2 ml staining buffer and centrifuge (500 x g, 5 minutes). 9. Add secondary antibody 5 g/ml in 100 l staining buffer. Incubate 30 minutes on ice in the dark. 10.Wash cells in 2 ml staining buffer and centrifuge (500 x g, 5 minutes). 11.Resuspend at 2 x 106/ml in cell buffer and load on chip according to the reagent kit guide.
Conclusion
Apoptosis is a critical mechanism for organisms to maintain tissuehomoeostasis. In this Application Note we showed that the Agilent 2100 bioanalyzer is a versatile tool to detect apoptotic cells. Protocols and a list of recommended reagents for detection of two apoptotic markers are given. Excellent reproducibility of results from different chips for both the Annexin V and the caspase- 3 assay is demonstrated. Data from the 2100 bioanalyzer compares very well with that of a conventional flow cytometer in spite of a 5-fold lower cell consumption. Data acquisition is done automatically and data analysis is achieved by an intuitive software package which does not require manual setting of instrument related parameters.

'"/>

Source:


Page: All 1 2 3 4 5 6 7 8 9
Related biology technology :

1. RT-PCR Primers for the Study of Apoptosis
2. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
3. Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays
4. Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar
5. Monitoring transfection efficiency by green fluorescent protein (GFP) detection with the Agilent 2100 bioanalyzer
6. Sensitive detection of tumor cells in peripheral blood of carcinoma patients by a reverse transcription PCR method
7. Rapid detection of genomic duplications and deletions using the Agilent 2100 bioanalyzer
8. Mutation detection for the K- rasand P16 genes
9. Multiplex protein detection with the ECL Plex fluorescent Western blotting system using the Ettan DIGE Imager
10. The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists
11. Front-face detection for high-concentrate, opaque or solid sample