This test employs the property of Annexin V:Biotin to bind to the membrane phospholipid phosphatidylserine (PS) in the presence of Ca2+. PS is exposed at the cell surface during the early stages of apoptosis. Detection of PS is a very sensitive method for detecting cells entering apoptosis, at a time point considerably ahead of nuclear changes such as DNA degradation.
The conjugation protocol used to prepare Annexin V:Biotin has not changed the native phospholipid binding properties of Annexin V. This protocol is designed to measure apoptosis easily and quickly in a sample of suspended cells.
Preservative Stabilisers
0.02%
Sodium Azide (NaN3)
1%
Bovine Serum Albumin
Reagents In The Kit
Annexin V:Biotin
1 x 0.5ml vial
Propidium Iodide
1 x 1.6ml vial at 20ug/ml
Binding buffer
1 x 50ml vial at x 4 concentrate
Note: This assay also requires streptavidin:FITC conjugate for visualisation (not supplied - see recommended useful reagents section).
Application
This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visitwww.abdserotec.com/protocols.
Application Name
Yes
No
Not Determined
Suggested Dilution
Flow Cytometry
Neat
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Store at +4oC. DO NOT FREEZE.
This product should be stored undiluted. Should this product contain a precipitate we recommend microcentrifugation before use.
1) Dilute the binding buffer 1:4 in distilled water (50ml binding buffer + 150ml distilled water).
2) Wash cells in PBS.
3) Resuspend cells in pre-diluted binding buffer, adjusting to a cell density of 2-5 x 105cells/ml.
4) Add 5ul Annexin V:Biotin to 195ul of the cell suspension prepared in step 3.
5) Mix and incubate for 15 minutes at room temperature.
6) Wash cells twice in 190ul of pre-diluted binding buffer.
7) Resuspend cells in 190ul pre-diluted binding buffer.
8) Add streptavidin:FITC conjugate.
9) Mix and incubate for 30 minutes in the dark, at room temperature.
10) Wash cells in 190ul pre-diluted binding buffer.
11) Resuspend cells in 190ul pre-diluted binding buffer.
12) Add 10ul of the Propidium Iodide solution.
13) Analyse by flow cytometry.
The flow cytometer is preferably set such that the Mean Fluorescence Intensity of the Annexin V 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 binding buffer at 2-5 x 105cells/ml. Proceed with step 2 as described above.
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