Staining Methods for Programmed Cell Death

  • Owen S. D. Wally
  • Claudio StasollaEmail author


Formation and maintenance of the body plan is determined by several intrinsic factors including cell division, differentiation, and death. While structural and molecular markers for the detection of division and differentiation have long been used, detection of dying or dead cells is often more difficult to accomplish. This is mainly due to the fact that cell death can be genetically programmed (programmed cell death, PCD) or unprogrammed, and this distinction is often difficult to capture. This protocol presents a simple staining technique using trypan blue that can be utilized as a preliminary study to determine the presence (or absence) of dead cells within the tissue analyzed, but without discriminating between the type of death (programmed or unprogrammed). Once the presence of dead cells is verified, the more expensive and time-consuming terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) analysis can be employed to localize cells undergoing PCD. Although straightforward in its steps, the presented protocol might require some optimizations depending on the type of the tissue used and/or its developmental stage.


Necrosis Programmed cell death Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) Trypan blue 


  1. 1.
    Schwartzman RA, Cidlowski JA (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14:133–151PubMedGoogle Scholar
  2. 2.
    Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald HR, Mannherz HG, Tschop J (1993) Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J 12:371–377PubMedCentralPubMedGoogle Scholar
  3. 3.
    Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12CrossRefPubMedGoogle Scholar
  4. 4.
    Hashimoto S, Koji T, Niu JZ, Kanematsu T, Nakane PK (1995) Differential staining of DNA strand breaks in dying cells by nonradioactive in situ nick translation. Arch Histol Cytol 58:161–170CrossRefPubMedGoogle Scholar
  5. 5.
    Gavrieli Y, Sherman Y, Bensasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501CrossRefPubMedGoogle Scholar
  6. 6.
    Gorczyca W, Gong JP, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945–1951PubMedGoogle Scholar
  7. 7.
    Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV, Lassmann H (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71:219–225PubMedGoogle Scholar
  8. 8.
    Gorczyca W, Bigman K, Mittelman A, Ahmed T, Gong JP, Melamed MR, Darzynkiewicz Z (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7:659–670PubMedGoogle Scholar
  9. 9.
    Wang H, Li J, Bostock RM, Gilchrist DG (1996) Apoptosis: a functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. Plant Cell 8:375–391PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Ning SB, Wang L, Song YC (2002) Identification of programmed cell death in situ in individual plant cells in vivo using a chromosome preparation technique. J Exp Bot 53:651–658CrossRefPubMedGoogle Scholar
  11. 11.
    Sloop GD, Roa JC, Delgado AG, Balart JT, Hines MO, Hill JM (1999) Histologic sectioning produces TUNEL reactivity—a potential cause of false-positive staining. Arch Pathol Lab Med 123:529–532PubMedGoogle Scholar
  12. 12.
    Kanoh M, Takemura G, Misao J, Hayakawa Y, Aoyama T, Nishigaki K, Noda T et al (1999) Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy—not apoptosis but DNA repair. Circulation 99:2757–2764CrossRefPubMedGoogle Scholar
  13. 13.
    Keogh RC, Deverall BJ, Mcleod S (1980) Comparison of histological and physiological responses to Phakopsora pachyrhizi in resistant and susceptible soybean. Trans Br Mycol Soc 74:329–333CrossRefGoogle Scholar
  14. 14.
    Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT (1999) The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth. Plant Cell 11:1695–1708PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Hamberg M, Sanz A, Rodriguez MJ, Calvo AP, Castresana C (2003) Activation of the fatty acid alpha-dioxygenase pathway during bacterial infection of tobacco leaves—formation of oxylipins protecting against cell death. J Biol Chem 278:51796–51805CrossRefPubMedGoogle Scholar
  16. 16.
    Gain P, Thuret G, Chiquet C, Dumollard JM, Mosnier JF, Burillon C, Delbosc B, Herve P, Campos L (2002) Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique. Br J Ophthalmol 86:306–310PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Department of Plant ScienceUniversity of ManitobaWinnipegCanada

Personalised recommendations