Cell Signaling Technology

Product Pathways - Apoptosis

PathScan® Apoptosis and Proliferation Multiplex IF Kit #7851

Products Included Product No. Volume Applicaton Dilution Reactivity
Primary Cocktail 7847 100 µl IF-IC, IF-P* 1:100 H, (Mk)
Detection Cocktail 7843 100 µl IF-IC, IF-P* 1:100 N/A
Kit Analytes Detection Dye Ex(max) (nm) Em(max) (nm)
Phospho-Histone H3 (Ser10) Alexa Fluor® 488 495 519
Cleaved-PARP (Asp214) Alexa Fluor® 647 650 665
α-Tubulin Alexa Fluor® 555 555 565

Applications Key:  IF-P=Immunofluorescence (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Apoptosis and Proliferation Multiplex IF kit offers a novel method to simultaneously monitor mitotic index and programmed cell death using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. This kit contains a cocktail of three high quality primary antibodies targeted against α-tubulin, phospho-histone H3 (Ser10), and cleaved PARP (Asp214), as well as a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody and dye pairings have been pre-optimized, and each kit contains enough reagents for 100 assays (based on a working volume of 100 μL/test).

Specificity / Sensitivity

α-Tubulin antibody detects endogenous levels of total α-tubulin protein. Phospho-Histone H3 (Ser10) antibody detects endogenous levels of histone H3 only when phosphorylated at Ser10. This antibody does not cross-react with other phosphorylated or acetylated histones. Cleaved-PARP (Asp214) detects endogenous levels of the large fragment (89 kDa) of human PARP1 protein produced by caspase cleavage. This antibody does not recognize full length PARP1 or other PARP isoforms.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine #9953 (right), using PathScan® Apoptosis and Proliferation Multiplex IF Kit. Red = α-tubulin, green = phospho-Histone H3 (Ser10), and blue = cleaved PARP (Asp214)

IF-P

IF-P

Confocal immunofluorescent analsyis of paraffin-embedded Rh30 xenograft, control (left) or rapamycin-treated (right), using PathScan® Apoptosis and Proliferation Multiplex IF Kit. Red = α-tubulin, green = phospho-Histone H3 (Ser10), and blue = cleaved PARP (Asp214).

Source / Purification

Monoclonal antibodies were produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3, a synthetic peptide corresponding to residues surrounding Asp213 of human PARP, or with full-length chicken α-tubulin purified from brain extracts.

Background

Apoptosis is a regulated physiological process leading to cell death. Initiator caspases cleave and activate downstream effector caspases that in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF, and lamin A, which induce apoptosis (1). Cleavage of the nuclear poly (ADP-ribose) polymerase PARP occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,3). PARP helps to maintain cell viability by playing key roles in many cellular processes, including DNA replication, repair, and recombination. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (4). Cell proliferation can be measured by studying the phosphorylation state of histone H3 and microtubule assembly during mitosis. Histone H3 is one of four distinct core histone proteins found in the nucleosome. Phosphorylation of histone H3 at Ser10, Ser28, and Thr11 is tightly correlated with chromosome condensation during both mitosis and meiosis (5-7). Heterodimers composed of α-tubulin and β-tubulin form globular tubulin subunits common to all eukaryotic cells. Tubulin polymers known as microtubules are fundamental cytosolic fibers important in mediating cellular movement, including meiotic/mitotic chromosome alignment, cytoplasmic membrane vesicle transport, and nerve-cell axon migration (8).

  1. Nicholson, D.W. (1999) Cell Death Differ 6, 1028-42.
  2. Lazebnik, Y.A. et al. (1994) Nature 371, 346-7.
  3. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
  4. Oliver, F.J. et al. (1998) J Biol Chem 273, 33533-9.
  5. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
  6. Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
  7. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
  8. Westermann, S. and Weber, K. (2003) Nat Rev Mol Cell Biol 4, 938-47.

Application References

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Protocols

* Product-specific protocol.

Companion Products

*IF-P protocol recommended unmasking buffer: CitrateThe transfer of the secondary cocktail contained in this kit is contingent on the buyer using the purchased product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity), for immunocytochemistry, high content screening (HCS) analysis, or flow cytometry applications. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.

com. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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