Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

He, Liusheng; Wu, Xiaoli; Meylan, Françoise; Olson, Douglas P.; Simone, James; Hewgill, Derek; Siegel, Richard M.; Lipsky, Peter E.

doi:10.1016/s0002-9440(10)63751-0

Cited by 37 publications

(33 citation statements)

References 49 publications

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“…2A, row 1), indicating that a subset of cells appears to have spontaneous activation of both caspase 3 (DEVDase, R3) and caspase 6 (VEIDase, R5). As previously observed with single caspase indicator constructs (28), these cells displayed minimal evidence of apoptosis as determined by annexin V-APC binding (data not shown). As expected, exposure to the DNA-damaging reagent, VP16, strikingly enhanced the activation of both DEVDase (R3, 80% compared to 29% in control) and VEIDase (R5, 66% compared to 23% in control) ( Fig.…”

Section: Optical Configuration For Measurement and Validation Of A 2-supporting

confidence: 85%

“…Cell culture and transfection for HeLa cells and Jurkat cells were described previously (28). MCF7 cells stably transfected with Fas/CD95 were described previously (37).…”

Section: Cell Culture Transfection and Chemical Treatmentmentioning

confidence: 99%

“…Caspase activity in these FRET-diminished cells was quantitatively calculated by the equation as previously described (28).…”

Section: Calculation Of Caspase Activity On Flow Cytometry Datamentioning

confidence: 99%

“…Previously, we and others described a flow cytometric optical configuration to visualize CFP fi YFP FRET and its application in monitoring basal and apoptosis-related caspase activity in living cells when a caspase-cleavage site was inserted between CFP and YFP (28,38,39). More recently, we have developed a 3-laser 6-color flow cytometric system to detect three distinct FRET events: CFP fi YFP FRET1, YFP fi mRFP FRET2, and 2-step-linked CFP fi YFP fi mRFP FRET (36).…”

Section: Optical Configuration For Measurement and Validation Of A 2-mentioning

confidence: 99%

“…Chemical substrates, including fluorogenic substrates and fluorochrome-labeled inhibitors of caspases (FLICAs), have been developed for this purpose (8,(21)(22)(23). More recently, genetically engineered probes have been described, consisting of a CFP-YFP fusion protein in which the fluorophores are separated by a very short FRET-permissive linker containing at least one caspase cleavage site, such as DEVD, LEVD, or another caspase-sensitive cleavage sequence (24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The linker is cleaved by activated caspases, triggering decreased FRET because of the resultant physical separation of CFP and YFP molecules.…”

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confidence: 99%

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Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Simone

Hewgill

et al. 2006

Cytometry Pt A

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Background: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. Methods: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. Results: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimula-

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Section: Optical Configuration For Measurement and Validation Of A 2-supporting

confidence: 85%

“…Cell culture and transfection for HeLa cells and Jurkat cells were described previously (28). MCF7 cells stably transfected with Fas/CD95 were described previously (37).…”

Section: Cell Culture Transfection and Chemical Treatmentmentioning

confidence: 99%

“…Caspase activity in these FRET-diminished cells was quantitatively calculated by the equation as previously described (28).…”

Section: Calculation Of Caspase Activity On Flow Cytometry Datamentioning

confidence: 99%

Section: Optical Configuration For Measurement and Validation Of A 2-mentioning

confidence: 99%

mentioning

confidence: 99%

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Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Simone

Hewgill

et al. 2006

Cytometry Pt A

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Apoptosis (Programed Cell Death) Studied by Fluorescence Spectroscopy

Quiroz

Khanal

Pappas

2000

Encyclopedia of Analytical Chemistry

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Several analytical methods, many of which rely on fluorescence processes, have been developed to study apoptosis (programmed cell death). Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is therefore a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and developments in probe and fluorescent methods for assaying apoptosis are ongoing. There are several standard techniques, such as flow cytometry and confocal microscopy, for apoptosis study in cells. In addition, new techniques such as superresolution microscopy, multiphoton excitation, and single‐cell−single‐molecule spectroscopy are quickly emerging. This article explores several fluorescence approaches used in apoptosis studies as well as describes the mechanisms and hallmarks of the apoptotic cascade. As apoptosis plays a very important role in both healthy and diseased organism functions, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.

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Apoptosis (Programmed Cell Death) Studied by Fluorescence Spectroscopy

Martínez¹,

Khanal²,

Pappas³

2020

Encyclopedia of Analytical Chemistry

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Several analytical methods have been developed to study apoptosis (programmed cell death), many of which rely on fluorescence processes. Apoptosis is a highly regulated biological event and is a vital process that helps regulate tissue growth, normal cell turnover, immune response, and tissue development. However, diseases such as cancer and heart disease are associated with malfunctions in the apoptosis machinery. There is, therefore, a need to elucidate the processes of apoptosis induction and inhibition. Fluorescence assays continue to play a major role in apoptosis assays, and probe and method development are ongoing. There are several standard techniques such as flow cytometry and confocal microscopy for apoptosis study in cells. In addition, new techniques such as super‐resolution microscopy, multiphoton excitation, and single cell‐single molecule spectroscopy are quickly emerging. This updated article will explore several fluorescence approaches used in apoptosis studies as well as describe the mechanisms and hallmarks of the apoptosis cascade. Since apoptosis plays such an important role in both healthy and diseased organism function, the need to develop and apply sensitive analytical methods continues to be of the utmost importance.

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Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

Abstract: Apoptosis is characterized by a distinct series of morphological and biochemical changes that eventually result in the programmed death of cells. It plays an essential role in morphogenesis, host defense and homeostasis of all tissues, including the immune system.

Cited by 37 publications

References 49 publications

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

Apoptosis (Programed Cell Death) Studied by Fluorescence Spectroscopy

Apoptosis (Programmed Cell Death) Studied by Fluorescence Spectroscopy

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