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Apoptosis Assays by Flow Cytometry


Apoptosis Assays by Flow Cytometry

Flow cytometry is a flexible and versatile method for measuring populations of cells undergoing apoptosis, or cell death, at different stages. Early in apoptosis, the mitochondria lose membrane potential and are unable to function properly, leading to cytochrome c release and the activation of caspases. Flow cytometry enables the detection of activated caspases in cells with the use of probe molecules containing a cleavage site specific for caspases 3 and 7.  When cleaved by these activated caspases in early apoptotic cells, the probe molecules enter the nucleus, bind to DNA, and fluoresce.

The early stage of apoptosis also involves phosphatidylserine (PS) translocation from the inner cellular membrane to the outer leaflet, exposing it to the extracellular environment.  During later stages of apoptosis, cellular membranes become permeable, compromising their integrity and selective transport of materials. Flow cytometry reagents such as annexin V can be used to bind exposed PS on cell membranes for labeling of early apoptotic cells, while the DNA binding dye 7-AAD can be used to label late apoptotic cells.

High-performance flow cytometers often require training or an extensive amount of experience to deliver high-quality results. Designed to meet the needs of the most demanding scientists while still being accessible for novice users, Agilent NovoCyte flow cytometers make it easy to preform apoptotic assays. Discover a line of flow cytometers that provides:

  • High-quality scatter resolution for small particle detection

  • Consistent results at varying flow rates

  • Accurate absolute cell counts, rendering reference beads unnecessary

  • Easy maintenance, automatic cleaning cycles



A Powerful Yet Affordable Flow Cytometer for Apoptosis Assays

NovoCyte flow cytometers are built with trusted Agilent quality and have a proven track record of delivering reliable performance. Avoid the hassle of manual maintenance or the need to routinely adjust your detector settings. An advanced fluidic design gives you the reproducibility you require, while also offering scheduled and automatic startup and shutdown, automatic cleaning cycles, and batch analysis and reporting. With a fast and robust autosampler, your flow cytometer can do the work for you and collect samples after you leave the lab. 


Detect Early and Late Apoptotic Cells using Annexin V and 7-AAD by Flow Cytometry


NovoCyte

This apoptosis assay shows that staurosporine induces apoptosis in both Jurkat T cells and HeLa cells, providing insight into the early and late stages of apoptosis. After staurosporine treatment for 4 hours, cells were analyzed for early and late apoptotic cells by staining for phosphatidylserine with annexin V-FITC and with the DNA binding dye 7-AAD. Cells were classified as early apoptotic with annexin V-only positive staining, and late apoptotic (or dead) cells with annexin V and 7-AAD positive staining.


Measure Mitochondrial Membrane Potential Loss in Early Apoptotic Cells


NovoCyte

Jurkat T cells are treated with staurosporine in this flow cytometric apoptosis assay, followed by the addition of the fluorescent dye JC-1. JC-1 is cell-permeable and aggregates inside mitochondria while membrane potential is maintained, emitting fluorescence in the PE channel. When the membrane potential is lost, JC-1 will not localize to mitochondria and will reside in the cytoplasm in its monomeric form, emitting fluorescence in the FITC channel. The ratio of PE to FITC fluorescence represents changes in the mitochondria membrane potential.

 

Using Flow Cytometry to Quantify Caspase 3 and 7 Activity in Early Apoptotic Cells


NovoCyte

This apoptosis assay shows staurosporine-treated Jurkat T cells exhibit more caspase activity than the untreated, control cells. Jurkat T cells were treated with 2 µM staurosporine for less than 1 hour or 4 hours and stained with cell‑permeable, DEVD‑conjugated fluorescent nucleic acid binding dye and 7-AAD dye. The untreated control cells show minimal caspase activity, while cells treated with staurosporine exhibit a significant increase in caspase 3 and 7 activity (99.7%). Apoptosis Assays by Flow Cytometry

 


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