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An update to DNA ladder assay for apoptosis detection.

Rahbar Saadat Y, Saeidi N, Zununi Vahed S, Barzegari A, Barar J - Bioimpacts (2015)

Bottom Line: 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.Primary and late apoptosis in the H2O2-treated cells was determined by flow cytometry analysis.This improved method is able to detect apoptosis in a cost effective/timely manner without need for commercial kits and special equipment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.

ABSTRACT

Introduction: 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.

Conclusion: 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.

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Mentions: Flow cytometry was applied to distinguish apoptotic and necrotic cell death. Apoptosis includes translocation of membrane phosphatidylserine (PS) from the inner side of the plasma membrane to the surface. Annexin V, a Ca+2-dependent phospholipid-binding protein, has high affinity for PS, cells were stained with Annexin V-FITC/PI.12,13 Our results demonstrated that 58.14% of H2O2- treated cells showed apoptosis in this cell line ( Fig. 2).


An update to DNA ladder assay for apoptosis detection.

Rahbar Saadat Y, Saeidi N, Zununi Vahed S, Barzegari A, Barar J - Bioimpacts (2015)

© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401164&req=5

Mentions: Flow cytometry was applied to distinguish apoptotic and necrotic cell death. Apoptosis includes translocation of membrane phosphatidylserine (PS) from the inner side of the plasma membrane to the surface. Annexin V, a Ca+2-dependent phospholipid-binding protein, has high affinity for PS, cells were stained with Annexin V-FITC/PI.12,13 Our results demonstrated that 58.14% of H2O2- treated cells showed apoptosis in this cell line ( Fig. 2).

Bottom Line: 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.Primary and late apoptosis in the H2O2-treated cells was determined by flow cytometry analysis.This improved method is able to detect apoptosis in a cost effective/timely manner without need for commercial kits and special equipment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.

ABSTRACT

Introduction: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus