Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry

Abstract

Background The fluorochrome‐labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308–313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. Methods Apoptosis of human MCF‐7, U‐937, or HL‐60 cells was induced by camptothecin (CPT) or tumor necrosis factor‐α (TNF‐α) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA‐specific fluorochromes was measured by LSC. Results Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF‐7 cells treated with CPT. FLICA binding was largely (>90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. Conclusions The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation. Cytometry 44:73–82, 2001. © 2001 Wiley‐Liss, Inc.

In vitro model of “wound healing” analyzed by laser scanning cytometry: Accelerated healing of epithelial cell monolayers in the presence of hyaluronate

“…At different time points after scratching of the monolayer, the fluorochrome-labeled inhibitor of caspases, FAM-VAD-FMK, was added to the cultures. FAM-VAD-FMK is an affinity ligand that reacts with active center of caspases (12)(13)(14). Its covalent binding to the cysteine at the active enzyme center occurs only after enzyme activation (12).…”

“…LSC also allowed us to relocate the measured cells for examination of their morphology by fluorescence microscopy (7,8). Apoptotic cells were identified on the scatter plots by their green fluorescence, evidence of caspase activation (12)(13)(14). Indeed, upon relocation and visual examination, these cells were smaller and had condensed, pyknotic nuclei, features characteristic of apoptosis (17,18).…”

“…At different intervals after inducing the scratch on the cell monolayer, as specified in the Results and figure captions, the medium was replaced with fresh medium containing the fluorochrome-labeled pan-caspase inhibitor, namely 20 M of FAM-VAD-FMK (Immunochemistry Technologies, Bloomington, MN) (12)(13)(14), and the cultures were transferred back into the incubator for an additional hour. The cells were then rinsed twice with PBS (2 min each) and fixed by immersing the slides in the Coplin jar containing 1% methanol-free formaldehyde (Polysciences, Warrington, PA) dissolved in PBS at room temperature for 15 min.…”

“…Cellular fluorescence was measured by LSC (CompuCyte, Cambridge, MA) using a 488-nm wavelength argon ion laser to excite the fluorescence and measure green (530 Ϯ 30 nm) and red (Ͼ590 nm) fluorescences by separate photomultipliers, as previously described (12)(13)(14). Because the x-y positions of the measured cells were recorded, the cells in areas on the microscope slide within approximately 250 m proximity to the scratch were measured separately and compared with the monolayer cells located far (Ͼ2 mm) from the scratch.…”

“…The scratch in a monolayer of renal epithelial cells was made as described in Materials and Methods. One hour later, the cultures were treated for an additional hour with 20 M of FAM-VAD-FMK, the carboxy fluorescein-tagged pan-caspase inhibitor (12)(13)(14). The cells were then fixed and their DNA was counterstained with propidium iodide in the presence of RNase.…”