Navigation Links
Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays

Introduction

The understanding of the cellular signaling processes leading to programmed cell death (apoptosis) is of utmost importance in the study of autoimmune diseases such as rheumatoid arthritis or Parkinsons and Alzheimers disease. Here, errors in the signaling cascade are believed to lead to premature apoptosis in the affected tissue. At the same time, damaged cells that do not enter the apoptotic pathway may proliferate unchecked and become a cancerous cell mass. Thus, the study of apoptosis benefits many different fields of medicine by offering multiple targets for specific drug treatment.

Caspases (cysteinyl aspartate-specific proteases) are a family of important signaling molecules with various tasks depending on subtype and organ. The activation of caspases is also a marker for cellular damage in diseases such as stroke or myocardial infarction (reviewed by Lavrik et al . 2005). Although the precise role in the initiation and progression of apoptosis is not known for all caspases, their involvement as an indicator alone and as a potential leverage point for drug treatment make them widely researched molecules.

We explored the use of the active form of caspase-3 for the detection of apoptotic events. This protease has been implicated as an effector caspase associated with the initiation of the death cascade and is therefore an important marker of the cells entry point into the apoptotic signaling pathway (Nicholson et al . 1995). Caspase-3 is activated by the upstream caspase-8 and caspase-9 and since it serves as a convergence point for different signaling pathways, it is well suited as a read-out in an apoptosis assay. Detection of active caspase-3 can be used in different cell lines or primary cells, does not require the use of transfection techniques and can be multiplexed with other probes to get an in-depth understanding of signaling events with cell-by-cell resolution. Since there have been reports that cells progressively undergo apoptosis when exposed to staurosprine over time (Jacobson et al . 1996), we investigated the time-dependence of apoptosis as measured by caspase- 3 activation. This application is also amenable for use in automated microscopes such as the BD Pathway Bioimager in a hands-off mode, making it useful for secondary screening applications or to gain an insight into apoptotic pathways using RNAi.

Methods

HeLa cells (ATCC, CCL-2, a human cervical cancer cell line) were maintained and grown in DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum. Cells were seeded in 96-well clear bottom tissue culture plates optimized for imaging applications (Cat. No. 353219) at 10,000 cells/well. After 16 hours in the incubator, staurosporine was added to the cells for 4 hours.

Staurosporine (Sigma-Aldrich, S4400) is a broad-spectrum kinase inhibitor known to activate the apoptosis pathway. To generate dose-response relationships, the drug was serially diluted in growth medium and equal volumes of a 2 final concentration was added to the wells; the final vehicle (DMSO) concentration did not exceed 0.5%. After incubation, cells were fixed with 3.7% formaldehyde (in PBS) for 1h at room temperature and methanol (100%) permeabilized for 10 min at 20C. Non-specific binding was blocked for 1h with 5% goat serum in PBS buffer containing 0.3% Triton X-100. Cells were then incubated with cleaved caspase-3 (Asp 175) rabbit polyclonal antibody (Cell Signaling Technology, 9661, 1:100 dilution) in 5% goat serum and 0.3% Triton X-100 over night at 4C. Further wash steps removed unbound antibody prior to the addition of the secondary Alexa 488 goat anti-rabbit antibody (Invitrogen, A11034, 10μL/mL) and Hoechst 33342 dye (5 μg/ml) in 5% goat serum and 0.3% triton X-100 for 1 hr at room temperature. The cells were then washed and imaged. For time course experiments, the drug treatment was staggered in hourly intervals and cells were fixed at the end of the experiment.

Images were acquired on the BD Pathway Biomager using the 20/NA 0.75 objective. The system allows great flexibility in the selection of the fluorescent dyes since it uses 2 lamps with broad illumination spectrum and a total choice of 16 excitation filters. Hoechst 33342 (Invitrogen, H3570) and Alexa Fluor images were acquired using standard filter sets for dyes in this range with careful consideration of bleed-through artifacts. The BD Pathway Bioimager also has sophisticated analysis software built in to acquire and analyze images with minimal manual adjustment. Caspase-3 is primarily located in the cytoplasm and the algorithm was set to sample an area around the nucleus as representative of the cytoplasm.

Results

Images acquired by the BD Pathway Bioimager showed a distinct labeling pattern of the activated caspase-3 in response to the apoptosis-inducing drug staurosporine ( Figure 1 ). The doseresponse relationship is highlighted in Figure 1 showing a screen capture of the acquisition software. Each thumbnail image shows a representative area of the sampled well.

The entry into the apoptotic pathway is terminal and once a cell commits, the caspase-3 pathway is completely activated. Therefore, the best measure of the relative apoptotic activity in a well is the percentage of cells above a certain threshold. The BD Image Data Explorer data analysis tool allows setting of multiple thresholds and to plot dose-response curves, calculate EC50 ( Figure 3 ) and Z using these percentages.

Discussion

Activation of the caspase-3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify activators and inhibitors of the death cascade. The response is both time and concentration dependent suggesting that multiple pathways play a role in triggering the caspase-3 activation. One could hypothesize that cells are most susceptible to staurosporine in a specific phase of the cell cycle and therefore over time, most cells will die, similar to the findings of Vermeulen et. al. (2003).

We show that this representative apoptosis assay can be used in an automated way using the BD Pathway Bioimager and its built-in image and data analysis tools making it amenable to a high-throughput application development. Our data supports findings by Jessel et. al. (2002) who report an EC50 for staurosporine of 200 nM. We specifically see the usefulness of this assay as a building block in a multiplexed high-content assay. This caspase-3 assay can easily be accommodated in a different fluorescence channel and since the BD Pathway Bioimager utilizes broad-spectrum lamps, many dye combinations are possible. The BD Pathway Bioimager and its software tools provide the optimal environment to conduct this kind of multidimensional analysis.

References

1. Jacobson, M.D., Weil, M., Raff, M.C.. 1996. Role of Ced-3/ICE-Family Proteases in Staurospirine-induced Programmed Cell Death. JCB , 133:1041-1051.

2. Lavrik I.N., Golks, A., Krammer, P.H.. 2005. Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115:10. 2665-72.

3. Nicholson D.W. Ali A., Thornberry N.A. et al . 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43.

4. Vermeulen, K., Berneman, Z.N., Van Bocksaele, D.R. 2003. Cell cycle and apoptosis. Cell Proliferation, 36:165-175.

5. Jessel, R., Haertel, S., Socaciu, C. Tykhonova, S., Diehl, H.A.. (2002). Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells. J.Cell.Mol.Med. 6. 82-92


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems
2. Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)
3. FLIPR Calcium 3 Assay Kit - An Optimal No-Wash Fluorescence Calcium Indicator Reagent for Non-Optimal Calcium Responses
4. Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation
5. RT-PCR Primers for the Study of Apoptosis
6. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
7. Apoptosis detection by annexin V and active caspase-3 with the Agilent 2100 bioanalyzer
8. Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar
9. An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis
10. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
11. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Caspase Activation Indicator Apoptosis Image Based Assays

(Date:7/30/2015)... 30, 2015 Ascendis Pharma A/S (Nasdaq: ... its innovative TransCon technology to address significant unmet ... a six-month Phase 2 study to evaluate the ... in 53 treatment-naïve, pre-pubertal children with growth hormone ... pleased with the top-line results from our Phase ...
(Date:7/30/2015)... (PRWEB) , ... July 30, 2015 , ... ... for advanced fluid applications and designed for continuous operation up to 1500 bar. ... sterilization-in-place, ensuring safety, efficiency, and preventing toxic contamination. , The Pony™ NS2006L homogenizer ...
(Date:7/29/2015)... July 29, 2015 US-Australian drug discovery company, ... confirmed it is committed to progressing its ground-breaking technology ... practicable and to ensure the Company delivers the best ... Iain Ross , said the Company currently had ... pre-clinical programs, discovery programs and academic partnerships and initiatives, ...
(Date:7/29/2015)... SAN FRANCISCO , July 29, 2015 /PRNewswire/ ... trialling mobile molecular testing for applications in food ... Ubiquitome,s hand-held, battery powered real-time PCR device, the ... the farm right through to the supermarket shelf ... world. This includes involvement in animal disease control ...
Breaking Biology Technology:Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 2Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 3Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 4Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 5Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 6Ascendis Pharma A/S Announces Positive Top-line Results from a Phase 2 Pediatric Study of Once-Weekly TransCon Growth Hormone for the Treatment of Growth Hormone Deficiency 7GEA Announces the Pony™ NS2006L… a Self-contained, High Pressure Lab Homogenizer for Product Development Ensuring Maximum Efficiency and Safety 2Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 2Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 3Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 4Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 5Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 6Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 7Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 8Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 9Newly Appointed Acting CEO Outlines Immediate Plans for Novogen 10AsureQuality and Ubiquitome Partner on Applications for Mobile Molecular Food Safety Testing 2
... promises of stem cells is the creation of new organs, grown ... organ donation. But experts expressed concerns at last week's Stem Cell ... cures do not materialize, and among investors if nobody can make ... stem cells could be used in the medical and pharmaceutical industries, ...
... also the corporate parent of U.S. Cellular, said its revenues for ... last year that it had to restate due to accounting problems. ... $43.3 million in the same quarter a year ago. , ,TDS ... from as early as 2000 through 2005. The process delayed the ...
... Wis. - Two weeks after solidifying its global market ... Thursday boosted its line of working capital as well. The ... funding from three venture capital sources. , ,Imago, a provider ... completed another round of funding with funding led by ...
Cached Biology Technology:Stem cells raise hopes, but is there money in them? 2Stem cells raise hopes, but is there money in them? 3Investors commit $3.4 million to Imago 2
(Date:7/21/2015)... , July 21, 2015  NXT-ID, Inc. (NASDAQ: ... biometric authentication company focused on the growing mobile ... wallet, announces that it has filed provisional patent ... Method. This invention highlights next ... authorizes an account, but also the user using ...
(Date:7/20/2015)... 2015  Acuity Market Intelligence,s latest research "The ... Commerce and Privacy" forecasts that between 2014 and ... be downloaded to smart mobile devices by 2.2 ... is projected to generate more than $67.9 billion ... forecast period.    "Biometrics is at ...
(Date:7/13/2015)... OXFORD, Conn. , Jul. 13, 2015 /PRNewswire/ ... the "Company"), a biometric authentication company focused on ... it has filed provisional patent 62/188684 for ... NXT-ID achieves another new convenient and secure method ... expands new, innovative payment methods, introduced with its ...
Breaking Biology News(10 mins):NXT-ID Patents Personalized Tokenized Payments for Next Generation Secure Payment Technology 2NXT-ID Patents Personalized Tokenized Payments for Next Generation Secure Payment Technology 3NXT-ID Patents Personalized Tokenized Payments for Next Generation Secure Payment Technology 4Biometrics on Smart Mobile Devices to Redefine Digital Identity with 12.9 Billion Biometric App Downloads between 2014 and 2020 2NXT-ID Patents Behavior-Directed Payments 2NXT-ID Patents Behavior-Directed Payments 3NXT-ID Patents Behavior-Directed Payments 4
... season coming up soon, egg consumption is expected to rise temporarily. ... of eggs they can? "Parasites such as fowl mites that ... more," says Bradley A. Mullens , a professor of ... to boost egg production is to rid the hens of these ...
... bacteria in the genus Rickettsia spread through a population of ... pest of global importance. Infected insects lay more eggs, develop ... to their uninfected peers. The discoveries were made by ... in the April 8 issue of the journal Science ...
... known to favor the evolution of a variety of traits, ... changes in metabolism and feeding behavior. Now researchers reporting online ... Press publication, have added sleeplessness to that list. "Cave-adapted ... Borowsky of New York University. "In some ways, their sleep ...
Cached Biology News:UC Riverside entomologists propose pesticide-free method to increase egg production 2Instant evolution in whiteflies: Just add bacteria 2Instant evolution in whiteflies: Just add bacteria 3Instant evolution in whiteflies: Just add bacteria 4Through evolution, cavefish have lost sleep 2
... The SYS203 is a complete, flexible ... in small samples, as is frequently ... is designed around Instechs miniature Clark-style ... an inch in diameter (3.2 mm), ...
... Cell Identification Kit. ALDEFLUORs ... substrate for the enzyme ... is highly expressed in ... making ALDH an extremely ...
... The GSTTag Monoclonal Antibody is a ... affinity to the 26 kDa glutathione-S-transferase ... This purified antibody is suitable for ... sequence expressed in E. coli , ...
... The MetaVue Imaging System is the cost-effective ... With commands tailored to common research imaging ... morphometric analysis and simple image analysis. ... control library means that MetaVue has the ...
Biology Products: