Apoptest Annexin A5-FITC kit (cat. no.:A700)
The A700 contains enough product to perform 200 tests/assays at a testconcentration of 125 ng/test to detect the early apoptotic cell. For routine apoptosis detection, 1000 tests/assays can be performed at a testconcentration of 25 ng/test.
Annexin A5-FITC Kit:
- 100 µl Annexin A5-FITC solution
- 10 ml 10 x concentrated buffer
- 250 µg solid Propidium Iodide
Contents
- 1 vial containing 100 µl Annexin A5-FITC solution.
- 6 vials containing 1.7 ml 10x concentrated binding buffer.
- 1 vial containing 250 µg solid propidium iodide
Features of Annexin A5-FITC
- Annexin A5-FITC : 1:1 stoichiometric complex.
- Purity : > 99% pure according to Fast Protein Liquid Chromatography.
- Quality : > 99% of the protein has full phospholipid binding properties according to ellipsometry.
- Quantity : 100 µl Annexin A5-FITC solution.
- Concentration : 250 µg/ml annexin
- Additives : None.
Biological properties
- Annexin A5-FITC binds to phosphatidylserine
Storage of Annexin A5-FITC solution and the 10 fold concentrated buffer.
- Store in the dark and refrigerated at 2-8°C.
Storage of the propidium iodide.
- Store in the dark and refrigerated at 2-8°C.
ApplicationThe APOPTEST™-FITC is designed to measure swiftly Apoptosis in a variety of suspended cell types by flow cytometry. Because there are no additives, that might interfere with cellular processes, the APOPTEST™-FITC can be applied for cell sorting of Apoptotic cells, which then can be further investigated without any interference by side-effects of additives.
The APOPTEST™-FITC is to be used in vitro for research purposes only, not for diagnostic or therapeutic procedures!
Click here for the A700 protocol
A700 protocol
The APOPTEST™-FITC employs the property of Annexin A5-FITC to bind to PS in the presence of Ca2+. Our conjugation protocol to form Annexin A5 with FITC of 1:1 stoichiometric complex has not changed the native phospholipid binding properties of Annexin A5. Binding kinetics show a fast association of Annexin A5-FITC with the phospholipid membrane if PS and Ca2+ are available. The APOPTEST™-FITC protocol is designed to measure easily and swiftly Apoptosis in a sample of suspended cells.
Materials
- APOPTEST™-FITC (Annexin A5-FITC solution and 10x concentrated binding buffer)
- Propidium Iodide (250 µg solid propidium iodide)
- Cells in suspension Ice
Apparatus
Method
- Dilute the 10x concentrated binding buffer 10 fold with distilled water and place the diluted buffer on ice.
- Dissolve propidium iodide in 1 ml de-ionised water (dH20) at a final concentration of 250 µg/ml.
- Wash the cells of interest with ice-cold culture medium or PBS and finally suspend them in ice-cold diluted binding buffer at 10?- 106 cells/ml.
- Dilute the Annexin A5-FITC solution 10 fold with cold diluted binding buffer.
- Add 5 µl diluted Annexin A5-FITC and 5 µl propidium iodide to 490 µl of the cell suspension prepared as given by step 3.
- Keep the tube on ice and incubate for 10 minutes in the dark.
- Then measure the cell sample by flow cytometry.
The flow cytometer is preferably set such that the Mean Fluorescence Intensity of the Annexin A5 negative population is between 1 and 10. Optimal parameter settings can be found using a positive control. For a positive control, incubate the cells with 3 % formaldehyde in buffer during 30 minutes on ice. Wash away the formaldehyde and suspend the cells in cold diluted binding buffer at 10?- 106 cells/ml. Proceed with step 4 as described above. The incubation with Annexin A5 and Propidium Iodide is formulated to be carried out on ice to arrest further progress of the cells through the stages of life => apoptosis => secondary necrosis. For rat thymocytes, we have shown that when kept on ice the population distribution (viable, apoptotic, secondary necrotic) remains stable for at least 6 hours.
Testconcentration |
25 ng/test |
125 ng/test |
Number of tests |
1000 |
200 |
Application |
Routine apoptosis measurement |
Early apoptosis measurement |