In healthy Jurkat cells, JC-1 formed aggregates in the mitochondria lead to a strong red fluorescent signal. Cells in which the mitochondrial potential was disrupted JC-1 did not form aggregates and therefore very little red fluorescence was observed.
Cellometer acquired multiple images per sample and the cell fluorescence intensities were exported and analyzed in FCS Express. The data was plotted as a histogram of the green and red fluorescence intensity. The decrease in the red/green mean fluorescence intensity (from 552.29 for control to 39.18 for treated) is an indication of the mitochondrial depolarization.
Detection of Caspase 3/8 Activity
Caspase 3/8 Overview
In non-apoptotic cells procaspase 8 and procaspase 3 are inactive cysteine proteases. Upon activation of the apoptosis, a signaling cascade occurs where procaspase-8 is cleaved to the active caspase-8 protease. Caspase-8 quickly converts procaspase-3 to active Caspase-3. Once caspase-3 is activated, a series of irreversible events are set in motion that lead to the death of the cell, including activation of the CAD (Caspase-Activated DNase) endonuclease that degrades DNA within the nucleus and initiates chromatin condensation.
The CaspGLOW™Fluorescein Active Caspase-3 or Caspase 8 Staining Kit offers a simple, sensitive method for detection of active Caspase-3/8 in living cells. When detecting caspase-3, we utilize a specific FITC-conjugated caspase-3 inhibitor, DEVD-FMK (or IETD-FMK for Caspase 8). Both inhibitors, DEVD-FMK and IETD-FMK, are cell-permeable and bind irreversibly to active caspase-3 or caspase 8 within the apoptotic cell. The fluorescent marker (FITC) on the inhibitors allows for the detection of cells undergoing apoptosis.
Cellular fluorescent intensity data is plotted as a histogram for each sample. The associated data table displays the percent of cells that are caspase (+) and the percent of the cell population that is caspase (–).
Caspase 3 Positive
Treated Jurkat cells were imaged and data exported to FCS Express Software. The mean intensity of the caspase signal was plotted as a histogram to determine the percent positive and percent negative of the treated Jurkat populations.