Apoptosis 1 Multiparameter Kit

Figure 1. L929 cells treated with 2.5 ?M paclitaxel for 30 hours (right), and untreated (left). F-actin appears in green (Alexa Fluor 488 Phalloidin), nuclei appear in blue (Hoechst), and mitochondria appear in red (Mito Tracker Red). Images acquired from the ArrayScan HCS Reader.

The assay is performed on live cells growing on standard high-density microplates. The Multiparameter Apoptosis 1 Reagent Kit, Thermo Scientific ArrayScan HCS Reader and the Multiparameter Apoptosis 1 BioApplication software provide for automated plate handling, focusing, cell image acquisition, analysis, and quantification of apoptosis.

Changes in the actin cytoskeleton have been reported as a potential parameter related to apoptotic changes based on observations of an early increase in F-actin content detected by labeling with phalloidin, an F-actin specific stain.  However, this correlation is not observed in all cases.  The F-actin content is determined by the amount of fluorescent phalloidin staining. Alexa Fluor 488-Phalloidin binding is proportional to the amount of F-actin present.

Changes in mitochondria are important for apoptosis.  Mitochondria release apoptogenic factors through the outer membrane and dissipate the electrochemical gradient of the inner membrane. This is thought to occur via formation of the mitochondria permeability transition.  Also, mitochondria can proliferate during stimulation of apoptosis, leading to an increase in total mitochondrial mass. Mitochondrial mass/potential changes are based on the uptake of MitoTracker Red into the mitochondria of live cells. Dye uptake is proportional to the membrane potential according to the Nernst equation. Measurements of MitoTracker labeling do not distinguish between alterations in mitochondrial potential and mass; however, if the number of mitochondria remains constant then the amount of dye is directly related to the membrane potential. If the number of mitochondria is not constant, then the signal will likely be dominated by the increase in mass.

Cells undergoing apoptosis generally exhibit one of two types of nuclear change: fragmentation or condensation.  During nuclear fragmentation, a circular or oval nucleus becomes increasingly lobular. Eventually, the nucleus dramatically fragments into multiple subnuclei. Nuclear condensation appears to arise as a consequence of the loss of structural integrity of the euchromatin, nuclear matrix and nuclear lamina.

 
Apoptosis 1 Multiparameter Kit Features
Multiplexed assay quantifies three features of apoptosis: nuclear morphology (condensation/ fragmentation), mitochondrial mass/potential, and cytoskeletal changes (f-actin content) specifically and reliably in whole cells
Quantification on a cell-by-cell basis; allowing direct cross-correlation of features
No cell lysis, purification, or filtration steps
Versatile assay is widely applicable to a variety of cell types
The Cellomics Multiparameter Apoptosis 1 Kits provide the reagents and optimized protocol for fluorescent staining and quantitative analysis of apoptosis by analysis of nuclear morphology, mitochondrial mass/potential and F-actin content. The kits include Hoechst Dye, Alexa Fluor 488-conjugated Phalloidin and MitoTracker Red to stain for the three respective parameters, each of which can be monitored on respective channels within the same population of cells.

   Product Detail 

The Multiparameter Apoptosis 1 HCS Reagent Kit addresses the multiplicity of apoptotic pathways by providing High Content Screening (HCS)-qualified fluorescent reagents and a validated, optimized protocol for simultaneous measurement of three fundamental parameters associated with the process of apoptosis: (1) nuclear morphology, (2) mitochondrial mass and/or membrane potential, and (3) changes in f-actin content.