Testing Data (Healthy cells fluoresce red,dying cells fluoresce green.Jurkat cells were stained withICT's MitoPT JC-1 dye andanalyzed with a fluorescencemicroscope containing a longband path filter (excitation at 490nm and emission >510 nm). Innon-apoptotic healthy cells (2cells at left), the reagent aggregates inside intact mitochondria and fluorescesred. As the mitochondrial membrane potential drops and cells enter apoptosis,the dye disperses throughout the cell. The reagent then assumes a monomericform and fluoresces green (3 cells on right).)
Testing Data #2 (Using a flow cytometer to analyze cells labeled with MitoPT JC-1, theinstrument will measure apoptosis by monitoring the amount of redfluorescence in each region. Healthy cells with intact mitochondria fluorescered due to aggregated MitoPT JC-1 and appear in R2. As the mitochondrialmembrane permeability collapses and cells enter apoptosis, MitoPT JC-1reagent is dispersed throughout the cell, converting to a green fluorescentmonomeric form. The amount of red fluorescence drops as these cells enterR3. In this example, Jurkat cells were either treated with DMSO (negative,non-induced cells) or with staurosporine (apoptotic, induced cells) for 4 hoursand then labeled with MitoPT JC-1 for 15 minutes.)
Testing Data (View a time-lapse movie of caspase-3/7 activation in cultured ratfibroblasts. Data courtesy of Dr. Martin Purschke, MA GeneralHospital. Windows Media Player or similar required to playthe.avi file.)
Testing Data #2 (Rat fibroblasts were seeded in a 12-well plate at 1x104 cells/mL and irradiatedthe following day. 23.1 uL of MR-(DEVD)2 solution was mixed with media andadded. 1 hour later, 300 uL media was added and cells MR-DEVD data.werephotographedover 16 hours.Bright field imagesshow increasingred intensityas caspaseactivity andapoptosisprogressed (Dr. Martin Purschke, Massachusetts General Hospital).)
Testing Data (ICT's TotalCytotoxicity Testcan be usedto easily analyzethe activity ofnatural killer(NK) cells. Basedon flowcytometric data,the formula[R2/(R2+R1)] X 100 was used tocalculate cytotoxicity. NK-killed necrotic cells are shown in the maroon line, andapoptotic cells are shown as the blue line. When added together, the total levelof cell death is calculated as the black line. In this example, cytotoxicityincreased as more NK effector cells were combined with target cells. As the ratioof Effector:Target-cells increased from 12.5:1 to 100:1 the percentage of targetcells dying increased from 11% to 60%.)
Testing Data #2 (As all target cellswere initiallystained green, theentire populationcan be counted inR3=R2+R1. After theeffector cells andred live/dead stainwere added, all thedead target cellswere stained red, shifting them up the Y-axis (R2), clearly distinguishing themfrom the live target cells (R1). (Live effector cells are at lower left, dead effectorcells are in upper left). Using the Basic Cytotoxicity Test, the analysis would becomplete (and R2 would be the maroon line above). However, ICT's TotalCytotoxicity Kit includes an additional caspase reagent to concurrently assessapoptosis (which is the blue line above). To complete the analysis using theTotal Cytotoxicity Test, further analyze R1 and R2 to isolate apoptotic cells.)