apoptosis detection using the acumen explorer™ ads and
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Apoptosis Detection Using the Acumen Explorer™ ADS and Promega's
Anti-PARP p85 Fragment AntibodyC e l l - B a s e d A s s a y
s
APOPTOSIS DETECTION USING THE ACUMEN EXPLORER™ ADS AND PROMEGA’S ANTI-PARP P85 FRAGMENT ANTIBODY
by Matthew Cook, Ph.D., Acumen Bioscience, and Terry Riss, Ph.D., Promega Corporation
Abstract Accurate detection and quantitation of apoptotic events are key to further understand cellular mechanisms with a view to discovering novel drugs. This study describes a method for the immunocytochemical detection of the 85kDa caspase-cleaved fragment of human PARP (a marker of apoptosis) in HeLa cells using the Acumen Explorer™ ADS instrument.
Introduction The Acumen Explorer™ High-Throughput Screening (HTS) and Assay Development System (ADS) are laser-scanning systems that exploit the power of multiplex fluorescence detection to allow monitoring of subtle changes in cell morphology and intracellular biochemical events. High- quality information can be easily obtained, making HTS and ADS instruments ideal for compound screening and assay development and hit-to-lead activities, respectively.
Poly (ADP-ribose) polymerase (PARP) is a nuclear DNA binding protein that detects DNA strand breaks and that functions in base excision repair (1). Following caspase- mediated cleavage, PARP no longer functions as a DNA repair enzyme, and the p85 Fragment can be used as a marker for early stage apoptosis (2).
Promega’s Anti-PARP p85 Fragment pAb(a) is a polyclonal antibody directed specifically against the 85kDa caspase- cleaved fragment (p85) of human PARP; it does not detect the intact 116kDa enzyme. The specificity of this antibody makes it an excellent tool for detecting early stage apoptotic events.
Staurosporine-Induced Apoptosis in HeLa Cells Methods for this assay were based on the Anti-PARP p85 Fragment pAb Technical Bulletin #TB273 (3). Briefly, each assay was performed in poly-L-lysine coated 96- or 384-well plates. HeLa cells (4,000 cells/well in 100µl in 96-well plates or 2,000 cells/well in 50µl in 384-well plates) were seeded and cultured for 16 hours. Cells were stimulated with a range of staurosporine concentrations for three hours and then washed once with PBS. Washing, fixing and antibody binding were carried out using the methods recommended in the Anti-PARP p85 Fragment pAb Technical Bulletin described above. Bound Anti-PARP antibody was detected with TRITC Affinipure F(ab´) 2 fragment goat anti-rabbit IgG (H+L) antibody (Jackson
Immuno Research Laboratories) at a final dilution of 1:500 from the 7µM stock solution (1mg/ml).
In this instance, plates were scanned in the Acumen Explorer™ ADS. This assay has been performed using the Acumen Explorer™ HTS instrument. The Acumen Explorer™ software allows the user to combine algorithms of choice from a software suite. In this way, assay applications can be developed to the user’s specifications, allowing detection and quantitation of fluorescent and morphological characteristics of every object in every well for multiple fluorescent emission wavelengths (Figure 1).
Data obtained using the method outlined in Figure 1 were plotted. Using the Anti-PARP p85 Fragment pAb, apoptotic events could be detected at staurosporine concentrations as low as 100nM (Figure 2).
A second protocol was applied in which a green fluorescent DNA stain (Syto®-16, Molecular Probes) was used to determine total cell count for each well. This permits normalization of data from well to well and allows valid comparison of data between wells. Total cell count data were obtained using Syto®-16 simultaneously with the detection of apoptosis using the Anti-PARP p85 Fragment pAb in a single scan with the Acumen Explorer™ ADS.
Data obtained from using this normalization method were plotted (Figure 3). Using the Anti-PARP p85
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Figure 1. Flow diagram illustrating the assay development process using the Acumen Explorer™ ADS.
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Fragment pAb, the limit of detection of apoptotic events was again 100nM staurosporine.
Summary The combination of Promega’s Anti-PARP p85 Fragment pAb and the Acumen Explorer™ fluorescence detection instrumentation provides an excellent method for the accurate and sensitive detection of apoptotic events in cells.
References 1. Trucco, C. et al. (1998) Nucl. Acids Res. 26, 2644–9.
2. Duriez, P.J. and Shah G.M. (1997) Biochem. Cell. Biol. 75, 337–49.
3. Anti-PARP p85 Fragment pAb Technical Bulletin #TB273, Promega Corporation.
Protocol
www.promega.com/tbs/tb273/tb273.html
Anti-PARP p85 Fragment pAb 50µl G7341
For more information about the Acumen Explorer™ HTS or ADS instruments, contact Acumen Bioscience Ltd. at Melbourn Science Park, Cambridge Road, Melbourn, Royston, Hertfordshire SG8 6EE, UK +44 1763 262233 or www.acumenbioscience.com (a)U.S. Pat. No. 6,350,452 and other patents pending. Explorer is a trademark of Acumen Bioscience Ltd. Syto is a registered trademark of Molecular Probes, Inc.
Figure 2. Detection of the 85kDa caspase-cleaved PARP fragment in staurosporine-treated HeLa cells. A dose response curve was obtained by plotting the number of apoptotic cells against the log concentration of staurosporine (µM).
Figure 3. Detection of the 85kDa caspase-cleaved PARP fragment in staurosporine-treated HeLa cells. A dose-response curve (red) was obtained by plotting cell number against the log concentration of staurosporine (µM). The green line indicates the values obtained for the no-antibody control.
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APOPTOSIS DETECTION USING THE ACUMEN EXPLORER™ ADS AND PROMEGA’S ANTI-PARP P85 FRAGMENT ANTIBODY
by Matthew Cook, Ph.D., Acumen Bioscience, and Terry Riss, Ph.D., Promega Corporation
Abstract Accurate detection and quantitation of apoptotic events are key to further understand cellular mechanisms with a view to discovering novel drugs. This study describes a method for the immunocytochemical detection of the 85kDa caspase-cleaved fragment of human PARP (a marker of apoptosis) in HeLa cells using the Acumen Explorer™ ADS instrument.
Introduction The Acumen Explorer™ High-Throughput Screening (HTS) and Assay Development System (ADS) are laser-scanning systems that exploit the power of multiplex fluorescence detection to allow monitoring of subtle changes in cell morphology and intracellular biochemical events. High- quality information can be easily obtained, making HTS and ADS instruments ideal for compound screening and assay development and hit-to-lead activities, respectively.
Poly (ADP-ribose) polymerase (PARP) is a nuclear DNA binding protein that detects DNA strand breaks and that functions in base excision repair (1). Following caspase- mediated cleavage, PARP no longer functions as a DNA repair enzyme, and the p85 Fragment can be used as a marker for early stage apoptosis (2).
Promega’s Anti-PARP p85 Fragment pAb(a) is a polyclonal antibody directed specifically against the 85kDa caspase- cleaved fragment (p85) of human PARP; it does not detect the intact 116kDa enzyme. The specificity of this antibody makes it an excellent tool for detecting early stage apoptotic events.
Staurosporine-Induced Apoptosis in HeLa Cells Methods for this assay were based on the Anti-PARP p85 Fragment pAb Technical Bulletin #TB273 (3). Briefly, each assay was performed in poly-L-lysine coated 96- or 384-well plates. HeLa cells (4,000 cells/well in 100µl in 96-well plates or 2,000 cells/well in 50µl in 384-well plates) were seeded and cultured for 16 hours. Cells were stimulated with a range of staurosporine concentrations for three hours and then washed once with PBS. Washing, fixing and antibody binding were carried out using the methods recommended in the Anti-PARP p85 Fragment pAb Technical Bulletin described above. Bound Anti-PARP antibody was detected with TRITC Affinipure F(ab´) 2 fragment goat anti-rabbit IgG (H+L) antibody (Jackson
Immuno Research Laboratories) at a final dilution of 1:500 from the 7µM stock solution (1mg/ml).
In this instance, plates were scanned in the Acumen Explorer™ ADS. This assay has been performed using the Acumen Explorer™ HTS instrument. The Acumen Explorer™ software allows the user to combine algorithms of choice from a software suite. In this way, assay applications can be developed to the user’s specifications, allowing detection and quantitation of fluorescent and morphological characteristics of every object in every well for multiple fluorescent emission wavelengths (Figure 1).
Data obtained using the method outlined in Figure 1 were plotted. Using the Anti-PARP p85 Fragment pAb, apoptotic events could be detected at staurosporine concentrations as low as 100nM (Figure 2).
A second protocol was applied in which a green fluorescent DNA stain (Syto®-16, Molecular Probes) was used to determine total cell count for each well. This permits normalization of data from well to well and allows valid comparison of data between wells. Total cell count data were obtained using Syto®-16 simultaneously with the detection of apoptosis using the Anti-PARP p85 Fragment pAb in a single scan with the Acumen Explorer™ ADS.
Data obtained from using this normalization method were plotted (Figure 3). Using the Anti-PARP p85
12 C E L L N O T E S I S S U E 4 2 0 0 2
Figure 1. Flow diagram illustrating the assay development process using the Acumen Explorer™ ADS.
Body Pages 8/30/02 1:01 PM Page 12
C E L L N O T E S I S S U E 4 2 0 0 2 13
C e l l - B a s e d A s s a y s
Fragment pAb, the limit of detection of apoptotic events was again 100nM staurosporine.
Summary The combination of Promega’s Anti-PARP p85 Fragment pAb and the Acumen Explorer™ fluorescence detection instrumentation provides an excellent method for the accurate and sensitive detection of apoptotic events in cells.
References 1. Trucco, C. et al. (1998) Nucl. Acids Res. 26, 2644–9.
2. Duriez, P.J. and Shah G.M. (1997) Biochem. Cell. Biol. 75, 337–49.
3. Anti-PARP p85 Fragment pAb Technical Bulletin #TB273, Promega Corporation.
Protocol
www.promega.com/tbs/tb273/tb273.html
Anti-PARP p85 Fragment pAb 50µl G7341
For more information about the Acumen Explorer™ HTS or ADS instruments, contact Acumen Bioscience Ltd. at Melbourn Science Park, Cambridge Road, Melbourn, Royston, Hertfordshire SG8 6EE, UK +44 1763 262233 or www.acumenbioscience.com (a)U.S. Pat. No. 6,350,452 and other patents pending. Explorer is a trademark of Acumen Bioscience Ltd. Syto is a registered trademark of Molecular Probes, Inc.
Figure 2. Detection of the 85kDa caspase-cleaved PARP fragment in staurosporine-treated HeLa cells. A dose response curve was obtained by plotting the number of apoptotic cells against the log concentration of staurosporine (µM).
Figure 3. Detection of the 85kDa caspase-cleaved PARP fragment in staurosporine-treated HeLa cells. A dose-response curve (red) was obtained by plotting cell number against the log concentration of staurosporine (µM). The green line indicates the values obtained for the no-antibody control.
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