Annexin V-Cy3 Apoptosis Kit Plus

Catalog #: K202 | abID: ab14144

Product Details

abID ab14144
Detection Method Flow cytometry using FL1 channel for SYTOX Green dye (Ex = 488 nm; Em = 530 nm) and FL2 channel for Annexin V-Cy3 (Ex = 543 nm; Em = 570 nm).
Species Reactivity Mammalian
Applications Detecting apoptosis in living cells by flow cytometry or fluorescence microscopy. Cy3 shows brighter red fluorescence.
Features & Benefits • Simple one-step procedure; takes only 10 minutes
• Fast and convenient
• Apoptotic cells show red fluorescence, dead cells show green fluorescence and • Live cells show little or no fluorescence.
Kit Components • Annexin V-Cy3
• SYTOX Green Dye
• Binding Buffer
Storage Conditions 4°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


The assay is based on the observation that soon after initiating apoptosis, cells 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 easily be 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 of fixation. The Annexin V-Cy3 Apoptosis Detection Kit Plus includes annexin V-Cy3, SYTOX green dye, and binding buffer. The SYTOX green dye is impermeant to • Live cells and apoptotic cells, but stains necrotic cells with intense green fluorescence by binding to cellular nucleic acids. After staining a cell population with annexin V-Cy3 and SYTOX Green dye in the provided binding buffer, apoptotic cells show red fluorescence, dead cells show green fluorescence and • Live cells show little or no fluorescence. These populations can easily be distinguished by Fluorescence microscopy using FITC and rhodamine filters or by flow cytometry using the FL1 channel (Ex. 488 nm/Em. 530 nm) for SYTOX Green dye and FL2 channel for Annexin V-Cy3 (Ex. 543 nm/Em. 570 nm).

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Can you use this kit with fixed cells?
Annexin V-cy3 will not bind to fixed cells. It needs to be applied on live cells. You can fix the cells post treatment.
The protocol in the datasheet for flow cytometry doesnt mention a wash step after Annexin/PI addition. can i confirm that this is correct?? I.e, 5min incubation then straight into flow cytometer
Typically washing is not needed before analysing in the flow cytometer. However, if you wish to fix your cells, this can be done after incubating with Annexin V in binding buffer. The cells can then be washed in PBS and fixed in 2% PFA for 15 mins.Once your cells have been re-suspended in PBS, you can also add a preservative/stabiliser if you are planning on keeping your samples for a long time. For example, 1 mg/ml BSA or 1% sodium azide.
I am looking to investigate cell apoptosis using 96 well plates. I was wondering if your Annexin V-Cy3 Apoptosis Detection Kit could be adapted to a 96 well plate fluorescent plate reader format?
This assay can be adapted to a plate reader format. For this particular assay, take 10^5 trypsinized cells into the plate wells, resuspend in 100 µl binding buffer, add 1µ Annexin V and PI respectively, incubate them in the dyes, give a gentle wash and then analyze. The cell number can vary from cell to cell and/or treatment protocol selected. Please take measures to prevent overcrowding in the wells and also aspirate solutions gently since the apoptotic cells tend to detach quickly..
Can you prelabel the cells and then follow apoptosis using this kit?
Unfortunately, the product has not yet been tested in this manner. We are unsure whether it might work and needs significant optimization with known controls and timepoints to determine if the kit is performing correctly in these conditions.
Can we use frozen samples with this assay?
This kit can only work on freshly prepared samples.
Can the kit work on bacteria or yeast cells?
The kit has been standardized for mammalian cells only
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Will trypsinizing the cells removed the phosphatidylserine?
The trypsin used in this kit should in no way affect the PS on the cell membrane.
Krols, Michiel et al. (2016) ER-Mitochondria contact sites: A new regulator of cellular calcium flux comes into play. J Cell Biol. 2016 Aug 15;214(4):367-70.
Morrow, M. et al.Stimulation of the Liver X Receptor Pathway Inhibits HIV-1 Replication via Induction of ATP-Binding Cassette Transporter A1. Mol. Pharmacol., 2010; 78: 215-225.
Boehm AL et al (2008) Mol. Pharmacol. 73: 1632-1642.
Lin K-R et al. (2007) J. Biol. Chem. 282: 21962-21972.
Sok J.C. et al. (2006) Clin. Cancer Res. 12: 5064-5073.
For more citations of this product click here