Question (12271) | Annexin V-PE-Cy5 Apoptosis Staining / Detection Kit (ab14159)

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Question

BATCH NUMBER 67413 ORDER NUMBER 76761 DESCRIPTION OF THE PROBLEM 1. Binding Buffer was not included in the kit. 2. When replaced by a buffer containing kit, antibody did not bind. There were no positive signal in the FACS analysis when using the positive (apoptotic cells) control. 3. No signals were detected in the experimental groups also. SAMPLE HEK 293 cells with or without serum withdrawal induced apoptosis for analysis in FACS PRIMARY ANTIBODY Annexin V-PG-Cy5 SECONDARY ANTIBODY No DETECTION METHOD FACS POSITIVE AND NEGATIVE CONTROLS USED 293 Cells with or without serum withdrawal induced apoptosis SAMPLE PREPARATION Cells were trypsined and washed in pbs for two times and then washed one time in Annexin binding buffer, then annexin V antibody was added, FACS were performed in 15 min. TRANSFER AND BLOCKING CONDITIONS Annexin V binding buffer, incubation 15 time RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? three HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes WHAT STEPS HAVE YOU ALTERED? Different dilutions of antibody had been tried and different positive control cells ( ok cells with ATP depletion, which has been shown previously induced apoptosis, therefore annexin V positive cells. ADDITIONAL NOTES The volume of the primary antibody (Annexin V-PG-Cys) is also less than what is indicated in the information sheet. Only about 50 -100 ul in the tube.

Answer

I'm very sorry to hear you are having problems with this kit and for the shortages you have experienced. I have arranged for 2 kits to be sent to you as soon as possible (order reference 80894) and apologise for the inconvenience. I also enclose the detailed protocol to help you, Please let me know if you need anything else and if you are satisfied with the new kits. Introduction: The Annexin V-PE-Cy5 Apoptosis Detection Kit is based on the observation that soon after initiating apoptosis, most cell types translocate the membrane phospholipid phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a strong natural affinity for PS. The one-step staining procedure takes only 10 minutes. In addition, the assay can be directly performed on live cells, without the need for fixation. Results can be analyzed by flow cytometry or fluorescence microscopy. II. Annexin V-PE-Cy5 Assay Protocol: A. Incubation of cells with Annexin V-PE-Cy5 1. Induce apoptosis by desired method. 2. Collect 1-5 x 105 cells by centrifugation. 3. Resuspend cells in 500 µl of 1X Binding Buffer. 4. Add 5 µl of Annexin V-PE-Cy5. 5. Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis. B. Quantification by Flow Cytometry Analyze Annexin V-PE-Cy5 binding by flow cytometry (Ex = 488 nm; Em = 670 nm). For analyzing adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-PE-Cy5 (A.3-5). C. Detection by Fluorescence Microscopy 1. Place the cell suspension from Step A.5 on a glass slide. Cover the cells with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on glass slide and visualize cells. The cells can also be washed and fixed in 2% formaldehyde before visualization. (Cells must be incubated with Annexin V-PE-Cy5 before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane.) 2. Observe the cells under a fluorescence microscope using the PE-Cy5 filter. Cells which have bound Annexin V-PE-Cy5 will show bright purple staining in the plasma membrane. III. Storage and stability: Store kit at 4oC.

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