Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis

Smolewski, Piotr; Grabarek, Jerzy; Halicka, H. Dorota; Darzynkiewicz, Zbigniew

doi:10.1016/s0022-1759(02)00074-1

Cited by 76 publications

(71 citation statements)

References 35 publications

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“…Because upon apoptosis induction cells asynchronously enter into and progress through the apoptotic process (43), the wider the detection time-window of the assay, the greater is its sensitivity (ability) to estimate the incidence of these apoptotic events. As shown in our previous studies (25), a combination of FLICA binding and the assay of plasma membrane integrity based on exclusion of PI reveals three distinct stages of apoptosis. This combination is of particular utility in discrimination between apoptosis and cell necrosis (44).…”

Section: Discussionsupporting

confidence: 59%

“…In our previous observations, we demonstrated that in all instances when cell death occurred by necrosis, without caspase activation, the cells did not bind FLICA (25,44). In the present study, we see a good correlation between caspase-3 activation and FAM-VAD-FMK binding among the early apoptotic cells that show condensed chromatin, but are not yet deficit in DNA content (Fig.…”

Section: Discussionmentioning

confidence: 74%

“…Their green and red fluorescence was measured by laser scanning cytometry (LSC) (30,31) using a 488-nm laser excitation. Other de tails of FLICA methodology are presented elsewhere (20,24,25 …”

Section: Flica Bindingmentioning

confidence: 99%

“…These reagents were designed as affinity ligands to react covalently with the reactive enzymatic center of activated caspases. During the past two years several articles have been published (19,(21)(22)(23), including the papers authored by us (18,24,25), in which these reagents have been used to probe for caspase activation. Based on a similar principle, other probes were developed to detect activation of intracellular serine proteases (26).…”

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confidence: 99%

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Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

et al. 2003

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BackgroundFluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.MethodsApoptosis of HL‐60, Jurkat, MCF‐7 and T‐24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H2O2). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase‐3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z‐VAD‐FMK or z‐DEVD‐FMK.ResultsFLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase‐3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM‐VAD‐FMK, FITC‐VAD‐FMK or FAM‐DEVD‐FMK binding to apoptotic cells could not be inhibited by z‐VAD‐FMK or z‐DEVD‐FMK, respectively, when the unlabeled inhibitors were added post‐induction of apoptosis.ConclusionsFLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases. Cytometry Part A 55A:50–60, 2003. © 2003 Wiley‐Liss, Inc.

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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 74%

Section: Flica Bindingmentioning

confidence: 99%

mentioning

confidence: 99%

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Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

et al. 2003

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“…Changes in cell size, shape, and granularity associated with apoptosis can be inferred from scattered laser light (7). Early intracellular events, such as the loss of the mitochondrial inner membrane potential or activation and the cleavage of caspases, can also be detected using electropotential sensitive dyes (8 -12) or fluorogenic substrates (13)(14)(15). Another early apoptotic event results in exposure of phosphatidylserine on the outer surface of the plasma membrane, which can be detected by fluorochrome-labeled annexin V (16 -19).…”

mentioning

confidence: 99%

Distinguishing modes of cell death using the ImageStream® multispectral imaging flow cytometer

et al. 2004

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Background: Here we demonstrate the ability of the ImageStream 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features. Methods: Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells. Results: Live (annexin V -, 7-AAD -) and early apoptotic (annexin V ϩ , 7-AAD -) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods. However, inspection of multispectral images of cells demonstrated that the annexin V ϩ , 7-AAD ϩ population contained both necrotic and late-stage apoptotic

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An ultra scale‐down methodology to characterize aspects of the response of human cells to processing by membrane separation operations

Masri

Lawrence

Wall

et al. 2017

Biotech & Bioengineering

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Tools that allow cost‐effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell‐based therapies. In this paper, an ultra scale‐down (USD) device has been developed for the characterization of the response of a human cell line to membrane‐based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line. The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. Transmission of LDH was investigated with time of operation and for the two disc speeds investigated (6,000 and 10,000 rpm or ϵ max ≈ 1.9 and 13.5 W mL−1, respectively). As expected, increased energy dissipation rate led to increased transmission as well as significant increases in rate and extent of cell damage. The method developed can be used to test the impact of varying operating conditions and cell lines on cell damage and morphological changes. Biotechnol. Bioeng. 2017;114: 1241–1251. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis

Cited by 76 publications

References 35 publications

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Distinguishing modes of cell death using the ImageStream® multispectral imaging flow cytometer

An ultra scale‐down methodology to characterize aspects of the response of human cells to processing by membrane separation operations

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