Annexin V-Biotin Apoptosis Staining / Detection Kit (ab14190)

Thank you very much for contacting us with your question.

We unfortunately do not sell the streptavidin-FITC to use with this kit, however we do have streptavidin-HRP available (ab7403). Would this be suitable for you?

Thank you for your enquiry. Annexin V staining is calcium dependent, therefore all buffers used after the annexin V staining step should contain calcium (1.8-2.5 mM). Fluorescence will fade gradually under the light, therefore any experiments performed after annexin V staining should be carried out in the dark. We have not done permeabilization here, however, double labeling of annexin V with a cell surface marker has been widely used successfully. If you have any additional questions, please contact us again.

The incubation time can be extended to 30 min. However, a longer incubation period can sometimes result in more dead cells, which can give false positive results. Generally you should limit it to 10 min. After incubation, you can put the cells on ice if you have to wait a while to do the next step. If you have any additional questions, please contact us again.

Thank you for your enquiry. All the information that we have regarding this product is located on the online datasheet. Here is the protocol, please let me know if you have any more questions.... Annexin V-Biotin Assay Protocol A. Incubation of cells with Annexin V-Biotin 1. Induce apoptosis by desired method. 2. Collect 1-5 x 105 cells by centrifugation. 3. Resuspend cells in 200 µl of 1X Binding Buffer. 4. Add 5 µl of Annexin V-Biotin and 5 µl of propidium iodide (PI, optional). 5. Incubate at room temperature for 5 min in the dark. 6. Wash the cells once in 200 µl of 1X Binding Buffer. Centrifuge to remove the buffer. 7. Fix cells with 2% formaldehyde in PBS for 15 min and wash cells once with PBS. Resuspend cells in 100 µl of PBS + 1 mg/ml BSA. Note: Cells must be incubated with Annexin V-Biotin before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane. 8. Add 5 µg/ml of avidin-fluorescein (not provided) and incubate for 15 min. 9. Collecting cells by centrifugation and resuspend in PBS. Proceed to B or C below depending on method of analysis. B. Quantification by Flow Cytometry Analyze samples by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually FL2). For adherent cells, gently trypsinize and wash cells once with serumcontaining media before incubation with Annexin V-Biotin (A.3-5). C. Detection by Fluorescence Microscopy 1. Place the cell suspension from Step A.9 on a glass slide. Cover the cells with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.9), invert coverslip on glass slide and visualize cells. 2. Observe the cells under a fluorescence microscope using a dual filter set for FITC & rhodamine. Cells that have bound Annexin V-Biotin and stained with (strept)avidine-FITC will show green staining in the plasma membrane. Cells which have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).