This page introduces methods for detecting apoptosis and links to relevant resources and papers.
In general, we have been looking at cell death in cultured adherant neuronal cells using confocal and laser scanning microscopy. We have initially used the TUNEL and Annexin techniques, there is a huge variety of techniques/analysis and applications used by different Labs.
Note also that many links on this page are to external internet resources.
Annexin
Caspase 3
Cleaved Actin
TUNEL
The TUNEL (TdT-mediated dUTP Nick-End Labeling) technique detects fragmented DNA, which occurs close to the final step in the apoptotic process.
Fragmented DNA of apoptotic cells can incorporate fluorescein-dUTP at 3Ç-OH DNA ends using the enzyme Terminal Deoxynucleotidyl Transferase (TdT), which forms a polymeric tail using the principle of the TUNEL assay. The labeled DNA can then be visualized directly by fluorescence microscopy or quantitated by flow cytometry.
Annexin V is an anticoagulant protein that preferentially binds negatively charged phospholipids. An early step in the apoptotic process is disruption of membrane phospholipid asymmetry, exposuring phosphatidylserine (PS) on the outer leaflet of the cytoplasmic membrane. Fluorescently conjugated Annexin V can be used to detect this externalization of phosphatidylserine on intact living cells. Propidium iodide is often combined as a second flurochrome to detect necrotic cells.
BD Biosciences Annexin V Staining Technique
R&D TACS Annexin V-FITC Apoptosis Detection Kitæ
Cat# TA4638,æAnnexin V, 100æTESTS
FITC Conjugation to Annexin Technique
Induction of apoptosis leads to procaspase-3 proteolytic cleavage to generate an active 18 kDa caspase-3 fragment which then targets key modulators of the apoptotic pathway including poly-ADP-ribose polymerase and other caspases, for cleavage.
procaspase-3 (32 kDa)
active fragment caspase-3 (18 kDa)
Several commercial suppliers have antibodies which recognize only the active 18 kDa fragment as a specific marker for apoptosis.
These commercial links are for educational purposes only and do not reflect endorsement of a specific product.
Trevigen Australian Distributor: Bio Scientific
Cat # 2305-PC-020, Anti-Cleaved Caspase 3, Rabbit Polyclonal Antibody, 20 µl
Cat # 2305-PC-100, Anti-Cleaved Caspase 3, Rabbit Polyclonal Antibody, 100 µl
Cell Signaling Technology Australian Distributor: Genesearch
Cat #9664, Cleaved Caspase-3 (Asp175) (5A1) Rabbit Monoclonal Antibody
BD Biosciences Australian Distributor: BD Biosciences Australia
Cat# 551150, Caspase-3, Active Form, Polyclonal CM1, Serum, WB, 100 µl
Cat# 559565, Caspase-3, Active Form, Monoclonal C92-605, Purified, FCM, IHC(Fr), IP, 25 µg
The C92-605 monoclonal antibody (mAb) specifically recognizes the active form of caspase-3 in human and mouse cells.
Chemicon Australian Distributor: CHEMICON Australia
Cat# AB3620, PARP Cleaved Form (Hu), Rabbit, 50 µg
Cat# AB3623, Cleaved Caspase-3, Active Form (Hu, Ms), Rabbit, 50 µg
Cat# AB3627, Cleaved Caspase-7, Active Form (Hu, Ms, Rt), Rabbit, 50 µg
Cat# AB3629, Cleaved Caspase-9, Active Form (Hu), Rabbit, 50 µg
OMIM - TUMOR NECROSIS FACTOR LIGAND SUPERFAMILY, MEMBER 6; TNFSF6 (FAS LIGAND; FASL APOPTOSIS ANTIGEN LIGAND 1; APT1LG1 APOPTOSIS ANTIGEN LIGAND CD95 LIGAND; CD95L CD178 ANTIGEN; CD178)
(More? Apoptosis - Fas Ligand)
Agar A, Yip SS, Hill MA, Coroneo MT. Pressure related apoptosis in neuronal cell lines. J Neurosci Res. 2000 May 15;60(4):495-503.
Adamec E, Yang F, Cole GM, Nixon RA. Multiple-label immunocytochemistry for the evaluation of nature of cell death in experimental models of neurodegeneration. Brain Res Brain Res Protoc. 2001 Jul;7(3):193-202.
Yang F, Sun X, Beech W, Teter B, Wu S, Sigel J, Vinters HV, Frautschy SA, Cole GM. Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease. Am J Pathol. 1998 Feb;152(2):379-89.
Urase K, Fujita E, Miho Y, Kouroku Y, Mukasa T, Yagi Y, Momoi MY, Momoi T. Detection of activated caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum. Brain Res Dev Brain Res. 1998 Nov 1;111(1):77-87.
Schmidt-Kastner R, Truettner J, Zhao W, Belayev L, Krieger C, Busto R, Ginsberg MD. Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat. Brain Res Mol Brain Res. 2000 Jun 23;79(1-2):88-101.
Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res. 2000 Aug 25;259(1):3.
de Boer RA, van Veldhuisen DJ, van der Wijk J, Brouwer RM, de Jonge N, Cole GM, Suurmeijer AJ. Additional use of immunostaining for active caspase 3 and cleaved actin and PARP fragments to detect apoptosis in patients with chronic heart failure. J Card Fail. 2000 Dec;6(4):330-7.
Telford WG, Komoriya A, Packard BZ. Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry. Cytometry. 2002 Feb 1;47(2):81-8.
Grabarek J, Amstad P, Darzynkiewicz Z. Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases. Hum Cell. 2002 Mar;15(1):1-12. Review.
Gown AM, Willingham MC. Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem. 2002 Apr;50(4):449-54
Kohler C, Orrenius S, Zhivotovsky B. Evaluation of caspase activity in apoptotic cells. J Immunol Methods. 2002 Jul 1;265(1-2):97-110. Review.
Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EA. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol. 2003 Feb;199(2):221-8.
Hartel S, Zorn-Kruppa M, Tykhonova S, Alajuuma P, Engelke M, Diehl HA. Staurosporine-induced apoptosis in human cornea epithelial cells in vitro. Cytometry. 2003 Sep;55A(1):15-23.
These commercial links are for educational purposes only and do not reflect endorsement of a specific product.
Promega Website http://www.promega.com/
Promega DeadEnd» Fluorometric TUNEL System
"The DeadEnd» Fluorometric TUNEL System measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP(a) at 3Ç-OH DNA ends using the enzyme Terminal Deoxynucleotidyl Transferase (TdT), which forms a polymeric tail using the principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay (1). The fluorescein-12-dUTP-labeled DNA can then be visualized directly by fluorescence microscopy or quantitated by flow cytometry."
Promega Notes Number 68, 1998, p. 13 Assay Systems for Detecting Apoptosis and Cell Death
Serotec Website http://www.Serotec.com/
Serotec product search results "apoptosis"
Serotec product search results "caspase"
Trevigen Website http://www.trevigen.com/
Australian Distributor Bio Scientific
Tel: +61 2 95 21 21 77 1 800 251 437 Fax: +61 2 95 42 31 00 email: info@biosci.com.au Web: http://www.biosci.com.au
Cell Signaling Technology Website www.cellsignal.com Australian Distributor: Genesearch
14 Technology Drive Arundel, Queensland 4214 Tel. Toll Free 1800 074 278 Fax (07) 5594 0562 E-mail: info@genesearch.com.au Website: www.genesearch.com.au
BD Biosciences Website http://www.bdbiosciences.com Australian Distributor: BD Biosciences Australia
BD - Australia / New Zealand 4 Research Park Drive Macquarie University, Research Park North Ryde, NSW, 2113 (612) 8875.7000
Chemicon Website http://www.chemicon.com Australian Distributor: CHEMICON Australia
CHEMICON Australia Pty. Ltd. 34 Wadhurst Drive Boronia Victoria 3155 Australia Phone: 3 9839 2000 Fax: 3 9887 3912 Email: custserv@chemicon.com.au Contact: Leanne Daly
There are many different methods for determining programmed cell death. This page covers only those intended for use in our Lab.
Please email Dr Mark Hill if you wish to make a comment about this current project.