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Over several decades, significant advances have been made in implementing several morphological and biochemical criteria to define and characterize apoptosis. In order to appropriately identify apoptosis in cellular cultures or in vivo (animal models), with the ultimate aim of discovering novel, useful, specific, and powerful pro-apoptotic antitumoral drugs, it is necessary to accurately validate apoptotic processes through morphological, biochemical, or immunological methods. Often, apoptosis is detected and/or measured by only one method and the authors frequently use imprecise terminology, such as ‘% apoptosis’, ‘% cell death’, instead of defining the specific method used such as ‘% cells with condensed chromatin’, ‘% cells TUNEL positive’, ‘% cells Annexin V positive’ and others.1 The Nomenclature Committee on Cell Death (NCCD) recommends and encourages researchers to demonstrate that apoptosis or other forms of cell death take place using more than one assay, as an artifact removal feature, and to avoid the ‘confusing and imprecise’ nomenclature, such as ‘% apoptosis’, which ‘should definitively be abandoned’.1 Moreover, the NCCD ‘urges’ all life science journals to join the Cell Death and Differentiation journal in adopting these recommendations.1 In order to determine the level of compliance with these recommendations, we analyzed 110 Cancer Research articles that detect/measure apoptosis, published between August 15, 2010 and February 15, 2011, and 120 similar research articles published in Cell Death & Disease between January 1, 2010 and September 2011. In 21 of the Cancer Research and 15 of the Cell Death & Disease articles, apoptosis was determined by the authors for different treatments or in different settings using a different combination of apoptosis techniques each time, thus leading the total number of apoptotic determinations (entries) to 137 (Cancer Research) and 136 (Cell Death & Disease) (see Table 1 and Table 2). The results clearly show that in only 60 of the 137 entries (43.8%) for Cancer Research and 96 of the 136 entries (70.58%) for Cell Death & Disease, the authors used at least two different methods for apoptosis detection. In addition, we determined that in 5 of the 137 (3.64%) entries from Cancer Research and in 34 of the 136 (25%) of the entries from Cell Death & Disease the authors use at least three methods for apoptosis detection (Figure 1). Moreover, it is important to emphasize that most, if not all of the apoptosis techniques used to detect apoptosis as single methods may, as shown by some reports, also detect necrosis or other cellular processes. As an example, DNA fragmentation measured by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) (26 of 137 from Cancer Research; 26 of 136 from Cell Death & Disease) or subG1 DNA content (30 of 137 from Cancer Research; 18 of 136 from Cell Death & Disease) or Annexin V alone, without PI/7AAD (16 of 137 from Cancer Research; 7 of 136 from Cell Death & Disease) and other assays can also detect necrosis.1, 2, 3, 4, 5, 6 In addition, caspases can be activated during cellular processes other than apoptosis.1 Thus, more than one method for apoptosis detection should be used. In order to investigate the proper use of the specific name for each method used, we analyzed the same entries described above. Our analysis shows similar results for the two journals. While for Cell Death & Disease, 83 from 136 entries (61.02%) use the name of the specific method used for apoptosis detection, among the 137 entries from Cancer Research, 87 (63.5%) use the name of the specific method used (Figure 1). These results certainly leave space for improvement. In conclusion, these results clearly suggest the need for improvements and of adequate guidelines for authors, reviewers, and editors regarding the apoptosis (in particular) and cell death (in general) detection, and quantification methods. The authors declare no conflict of interest. OB received a fellowship from the Lady TATA Memorial Trust, London, UK. This work was also supported by the National Institutes of Health grants CA127264 (to AA), CA105306, CA131664, and HL080192 (to RK).

Analysis of apoptosis methods recently used in Cancer Research and Cell Death & Disease publications