Quantitative comparison of in situ methods for detecting apoptosis induced by X-ray irradiation in mouse thymus

Apoptosis, or programmed cell death, is a process fundamental to the homeostasis of multicellular organisms. Therefore, the development of methods for detecting dying and dead cells is of great importance. In the present study, four methodologies for identifying thymocyte apoptosis were evaluated and compared: a classical Haematoxylin and Eosin staining method, two histochemical methods (DNA polymerase-mediated in situ end-labelling and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labelling) and flow cytometry. Aspects important for the quantitation of apoptosis in the mouse thymus after a low dose (below 1 Gy) of X-ray irradiation were emphasized. The nick-end-labelling method had the highest sensitivity among the four methods in detecting apoptotic cells; however, the in situ end-labelling method showed sensitivity similar to nick-end-labelling and possessed better response to incremental increases in radiation. The sensitivity of the Haematoxylin and Eosin staining was lower than the above two methods. Flow cytometry could not detect low-frequency apoptosis after a low radiation dose of below 30 cGy (cGy = 0.01 Gy) and did not respond linearly to increasing radiation. We conclude, therefore, that the in situ end-labelling method is the most adequate of the methods tested, especially for the detection of low-frequency apoptosis.