title: An update to DNA ladder assay for apoptosis detection.
abstract: BACKGROUND
  A growing interest in apoptosis, programmed cell death, in the last years is observed and leads to better understanding of molecular mechanisms during cell-cell signaling, cell-environment interaction and screening of drugs. This in turn results in emerging of new assays and development of more accurate kits for fast and early detection of apoptosis. However, their sensitivity and reliability have often been scrutinized. Here we introduce a rapid and improved method of DNA ladder apoptosis assay for evaluating apoptosis in mammalian cells.
  
  
  METHODS
  NIH-3T3 cell line was used in this study. After treatment of cells with apoptotic agent, 500 μM H2O2 at 48 hours, DNA was extracted. Then an update protocol of DNA ladder assay was applied for detection of apoptosis. Flow cytometry and DAPI staining were performed to verify apoptosis.
  
  
  RESULTS
  Primary and late apoptosis in the H2O2-treated cells was determined by flow cytometry analysis. DAPI Staining used to show DNA damage and DNA ladder assay using 1.5% gel electrophoresis showed fragmentation in the DNA of treated cells.
  
  
  CONCLUSIONS
  In this research we aimed to improve DNA ladder assay to the high quality detection of apoptosis in mammalian cells. In our strategy, employing a practical DNA extraction protocol, DNA ladder assay could be applied as an easy/fast method for apoptosis detection. This improved method is able to detect apoptosis in a cost effective/timely manner without need for commercial kits and special equipment.
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Yalda
authors.initials: Y
authors.lastname: Rahbar Saadat
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Nazli
authors.initials: N
authors.lastname: Saeidi
authors.affiliation: Student Research Committee, School of Advanced Biomedical Sciences, Tabriz University of Medical Science, Tabriz, Iran.
authors.forename: Sepideh
authors.initials: S
authors.lastname: Zununi Vahed
authors.affiliation: Student Research Committee, School of Advanced Biomedical Sciences, Tabriz University of Medical Science, Tabriz, Iran.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Barzegari
authors.affiliation: Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Jaleh
authors.initials: J
authors.lastname: Barar
keywords: Apoptosis; Cancer; DNA extraction; DNA ladder assay; Signaling pathway