The degree o apoptosis in a cell population is an importantparameter that contributes to a comprehensive pictureo cell health. Accurate detection and measurement o cellular apoptosis is essential or drug development anddiscovery, or understanding mode o compound action,and or understanding the impact o culture and growthconditions. However, the assessment o cellular apoptosishas been limited due to the requirements or expensiveand complicated instrument platorms, expertise andimproved analytical methods that provide rapid, robust andreproducible apoptosis data. Access to these improvementscan acilitate apoptosis monitoring, thereby enabling theecient, daily execution o cellular research.The Muse™ Cell Analyzer is a novel instrument that enablesmultidimensional cell health analysis on a single platorm.This small, benchtop cell analyzer eortlessly guides usersthrough the acquisition and analysis o samples usinga highly simplied and intuitive touchscreen interacewhich delivers rapid measurements o cell concentration,viability, apoptotic status, and cell cycle distribution
. Usingmultiparametric fuorescent detection o individual cellsvia microcapillary fow technology, the system enableshighly sensitive and rapid detection o cellular samplesusing minimal cell numbers. The simplied ormat enablesresearchers o varying backgrounds and experience levelsto obtain a comprehensive picture o cellular health.In this study, we used the Muse™ Cell Analyzer orapoptosis studies using the Muse™ Annexin V & DeadCell Assay, a rapid, no-wash assay or the identicationo apoptotic cells. Jurkat and HeLa cells were treated witha range o cytotoxic and anti-tumor compounds, and theresults rom percentage o live, early, late apoptotic, anddead cells were evaluated. Our results demonstrate thatthe assay provides rapid and sensitive detection o cellularapoptosis and provides quantitative and precise results ona variety o cellular types and compound treatments.
Maeials an Mehs
Apoptosis, or programmed cell death, is an importantregulator o cell growth and prolieration and ischaracterized by distinct morphological changes. Onekey early aspect o apoptosis is the translocation o phosphotidylserine rom the inner to the outer leafet o the plasma membrane and exposure to outer surace o thecell. This universal phenomenon is independent o species,cell type and induction system and occurs early in theapoptotic process.The Muse™ Annexin V & Dead Cell Assay is based on thedetection o phosphatidylserine (PS) on the surace o apoptotic cells and uses a premixed reagent containingfuorescently labeled Annexin V in combination with adead cell marker, 7-AAD. Annexin V is a Ca
-dependentphospholipid-binding protein that has a high anity orPS, a membrane component normally localized to theinternal ace o the cell membrane. Early in the apoptoticpathway, molecules o PS are translocated to the outersurace o the cell membrane where Annexin V can readilybind to them (Figure 1). Late-stage apoptotic cells showloss o membrane integrity. The membrane-impermeantdye, 7-AAD, is used to distinguish dead cells rom earlyapoptotic cells. The assay can thus distinguish ourpopulations:
Viable cells, not undergoing detectable apoptosis:Annexin V (–) and dead cell marker (–)
Early apoptotic and dead cells: Annexin V (+) and deadcell marker (–)
Late apoptotic or dead cells: Annexin V (+) and dead cellmarker (+)
Cells that have died through non-apoptotic pathway:Annexin V (–) and dead cell marker (+)
Simlied evaluatin atsisusing the Muse
Asima Khan, Kaheine Gillis, Jlie cl, Kamala tyagaajan
EMD Millire Crratin
The cmbineduse furescent labeledAnnexin V and membrane-imermeant 7-AAD DNA-binding dye can distinguishlive, early attic, and lateattic cells.
Healthy CellApoptotic CellLate Apoptotic Cell