Table of Contents
What is annexin V PI staining?
The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5.
What are the hallmarks of apoptosis?
Apoptosis is characterised by a series of typical morphological features, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells.
What is PI in flow cytometry?
Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.
How can you detect early and late apoptotic cells by flow cytometry?
Their logarithmically amplified green and red fluorescence signals were measured by flow cytometry. Live cells (V) are both FAM-VAD-FMK and PI negative. Early apoptotic cells (A) bind FAM-VAD-FMK but exclude PI. Late apoptotic/secondary necrotic cells (LA) are both FAM-VAD-FMK and PI positive.
Can I use PE and PI together?
PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE).
What are the markers used to detect apoptosis?
The markers most commonly used for in vitro detection of apoptosis include caspase-3/7 activity and phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane.
How is apoptosis detected in a multimode plate reader?
A variety of assays are available to detect apoptosis; however, few are conveniently adapted for high-throughput screening (HTS) using a multimode plate reader. The markers most commonly used for in vitro detection of apoptosis include caspase-3/7 activity and phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane.
How do you measure apoptosis?
There are a number of methods for running an apoptosis assay to measure these markers of apoptosis. Other assay methods are used to assay necrosis, anoikis and autophagy. To further investigate the up and downstream signaling pathways relevant to apoptosis we offer a panel of CRISPR engineered KO cell lines.
Is caspase activity a marker of apoptosis?
Introduction Caspase Activity Detection Protocol to Detect Caspase-3/7 Activity as a Marker of Apoptosis Detection of PS Exposure as a Marker of Apoptosis Protocol to Detect PS Exposure as a Marker of Apoptosis Data Calculation Commercial Reagents Conclusion References