HL-60 cells were treated with camptothecin, an apoptosis inducer, for 3 hours then stained with red SR-FLICA poly-caspase inhibitor SR-VAD-FMK and green FAM-FLISP serine protease inhibitor FFCK for 1 hour. Cells were washed, then analyzed on a scanning laser cytometer. Cells with active caspases stain red with SR-VAD-FMK along the X-axis and cells with active serine proteases stain green with FFCK along the Y-axis. Co-localization of caspase activity versus serine protease activity is evident in dually stained cells (B, C, D, and G). The light-scatter image (F) reveals many negative cells. In this experiment, the treatment triggered both caspase activity and serine protease activity. Activation of caspases was rapidly followed by serine protease activation and the time between was relatively short as very few cells are just red (G).
Jurkat cells were grown in suspension to 4x10e5 cells/ml, then divided into two separate TC-flasks. One population, “Non-Induced”, received a DMSO vehicle control (A). The other population, “Induced”, was spiked with 1uM staurosporine (B). Cells were incubated for 4 hours at 37°C, then stained with red SR-FLICA caspase-8 inhibitor, SR-LETD-FMK for 1 hour at 37°C. After labeling, samples were washed three times and slides were prepared. Fluorescence images were acquired using a Nikon Eclipse 90i microscope equipped with a Hamamatsu Flash 4.0 camera. In the treated sample, cells appear bright red, indicating a high level of caspase-8 activity (B, Induced, right). In the non-induced sample, few red positive cells are visible, indicating minimal caspase-8 activity (A, Non-Induced, left).
Poly-caspase SR-FLICA reagent, SR-VAD-FMK, was used with 7-AAD, a red live/dead stain, to simultaneously assess apoptosis and necrosis in K-562 human erythroleukemia cells. Cells were grown to 3x10e6 cells per sample and treated with a condition which induced apoptosis. Cells were stained with SR-VAD-FMK, washed, stained with 7-AAD, and analyzed using bi-color flow cytometry. A dot plot was set up to detect caspase activity (orangy-red, FL-2) on the X-axis and necrosis (red, FL-3) on the Y-axis, and carefully gated to distinguish SR-FLICA and 7-AAD fluorophores within a single sample tube. Four populations of cells were detected: unstained live cells have minimal fluorescence (67%, lower left); cells in early apoptosis fluoresce orangy-red with SR-FLICA (8%; lower right); cells in late apoptosis are dually stained with SR-FLICA and 7-AAD: they fluoresce orangy-red (they have active caspases) and red (the cell membrane has permeabilized) (23%; upper right); and necrotic cells fluoresce red with 7-AAD (2%; upper left).
Alzheimer’s disease (AD) patients exhibit plaques of amyloid beta (Abeta) in the brain. In healthy people, Abeta is phagocytosed and cleared from the brain by macrophages. It is proposed that AD may be caused by a malfunction of the macrophage, which prevents them from properly clearing Abeta. Curcumin, a substance found in tumeric, is thought to enhance the function of macrophages. SR-FLICA was used to test this theory in AD macrophages. Untreated control AD macrophages and curcumin-treated AD macrophages were exposed to FITC-labeled Abeta and stained with red SR-DEVD-FMK Caspase-3/7 SR-FLICA BioAssay™ kit. Macrophages that engulf FITC-Abeta will fluoresce green (right), and caspase-positive cells undergoing apoptosis will fluoresce red (left). In this experiment, essentially all of the untreated AD macrophages engulfed FITC-Abeta (green, top right) and became apoptotic (red, top left). They are dually stained green and red. Curcumin-treated AD macrophages engulfed FITC-Abeta (green, bottom right) but did not become apoptotic, as evidenced by the lack of red fluorescence (bottom left). It appears that in some AD patients, macrophages engulf Abeta but undergo apoptosis before Abeta is cleared from the brain. Curcumin may protect macrophages from apoptosis; more studies are needed to validate this conclusion.
Supplier Page from United States Biological for SR FLICA® Caspase 8 BioAssay™ Kit