On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Abstract

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3'-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3'-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles

“…DNase I treatment conditions (15 U, 35 minutes at room temperature) used in this study resulted in DAPI‐stained nuclei in some cells. This result is comparable with other studies using similar DNase treatment conditions (Fox, Parks, & Dutch, ; Lebon et al, ; Zhang et al, ).…”

Downregulation of DEC1 contributes to the neurotoxicity induced by MPP+ by suppressing PI3K/Akt/GSK3β pathway

“…The reason for selecting the subacute mouse model of PD is that it yields a more progressive neurodegeneration with apoptotic cell death than acute mice model . Based on the fact that DEC1 is an anti‐apoptotic factor, the reduction in DCE1 plays a determinant role in MPP + ‐induced neurotoxicity. We have several lines of evidence to support this notion.…”

“…For TUNEL assay, TUNEL‐positive cells were determined as dead cells. At least 400 cells from three randomly selected fields were counted for each dish …”

Identification of Cell-Free DNA Methylation Patterns Unique to the Human Left Ventricle as a Potential Indicator of Acute Cellular Rejection

“…Finally, the TUNEL assay that we used to assess cell death is non-specific and labels all fragmented DNA and includes those cells undergoing either apoptosis or necrosis. 30, 31 However, both apoptosis 25 and necroptosis 32, 33 are involved in acute and chronic cardiac allograft rejection. In the future, it will be useful to evaluate phosphorylated mixed lineage kinase domain-like protein (pMLKL) in our tissue sections to assess the contribution of necroptosis to cell death along with a more specific marker for apoptosis (e.g.…”