NC-3000™ Annexin V Assay

– For easy estimation of apoptosis

Acquisition, analysis and data presentation in one simple step

Translocalisation of phosphatidylserine (PS) from the inner to the outer membrane layer happens early in apoptosis and precedes other apoptotic events such as loss of membrane integrity and DNA fragmentation. Annexins are group of cellular proteins that bind to phospholipids in a calcium-dependent manner. A member of this group, Annexin V, has proven to be a useful tool in detecting apoptotic cells since it has a high affinity for PS. Using fluorescently labeled Annexin V the externalization of PS can easily be detected. Annexin V may also bind to PS on late apoptotic and necrotic cells but as the membrane integrity on these cells has been lost, these cells can be distinguished from early apoptotic cells by the use of the impermeant dye propidium iodide.

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Using fluorescence microscopy and image analysis the NC-3000 system automates detection of apoptotic cells based on phosphatidylserine externalization. Cells are stained with Hoechst 33342, PI and FITC labeled Annexin V.

Hoechst 33342 stains the total cell population, while Annexin V stains apoptotic and necrotic cells. Early apoptotic cells exclude PI, while late stage apoptotic and necrotic cells stain positively for both Annexin V and PI.

Jurkat cells stained with Hoechst-33342, Annexin V FITC conjugate and propidium iodide (PI). Cells were grown in the absence (image 1) or in the presence of 10 µM camptothecin (CPT) for 5 hours at 37°C (image 2). Cells were stained and analysed on NC-3000 using the Annexin V protocol. In this example  2% of the untreated Jurkat cells are early apopotic, whereas 40% of the CPT treated Jurkat cells are in early apoptosis. Lower panel shows the images acquired for the two samples.

Key benefits

         

Documentation

Product page NC-3000
Datasheet
References