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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
Cell Dissociation Enzymes Affect Annexin V/Flow-Cytometric Apoptotic Assay Outcomes After miRNA-based Transient
Christina M Gobin1, James N Menefee1, Chayil C Lattimore1
1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, U.S.A.
Background/Aim:
miRNA functional analysis involves transfection with miRNA-based oligos to identify gain-of or loss-of function cellular phenotypes. Apoptosis is a common phenotypic endpoint for miRNA functional analysis. We report that four common cell dissociation enzymes, TrypLE, Accutase, Trypsin, and Accumax, can differentially impact cell viability and apoptosis in Annexin V flow cytometric analysis after miRNA-based transient transfection.
Materials And Methods:
We transiently transfected a nonsense oligo into an epithelial cancer cell line (UM-SCC-12) for 24 h. Cells were harvested with either TrypLE, Accutase, Accumax, or Trypsin after 5 min. The Annexin V/7-AAD assay via flow cytometry was employed. Studies were performed in triplicate. Significant effects were detected by ANOVA, followed by Tukey's Multiple Comparison tests.
Results:
Trypsin produced the lowest cell viability and lowest percentage of apoptotic cells, specifically when compared to TrypLE and Accutase, respectively (p<0.01). Importantly, transfected trypsinized cells had a significant difference in cell viability and necrosis (p<0.05) when compared with non-transfected trypsinized cells, highlighting the influence of miRNA-based transfection on Annexin V flow cytometric outcomes. Interassay variability was lowest with TrypLE (1.13 %). As such, TrypLE provided the greatest reproducibility and reliability in our cell line.
Conclusion:
Our study highlights the variable effects of cell dissociation enzymes on transfected cells. Overall, the variability may lead to errors in detection of apoptotic cells using the Annexin V assay after miRNA-based transfection. Before assay use, we recommend pretesting cell dissociation enzymes on transfected cells to ensure reliable and reproducible results.
Insights
Cell dissociation enzymes significantly impact miRNA transfection results in apoptosis assays. Pretesting enzymes like TrypLE, Accutase, Trypsin, and Accumax is crucial for reliable miRNA functional analysis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- MicroRNA (miRNA) functional analysis typically involves transfecting cells with miRNA-based oligonucleotides (oligos) to observe cellular phenotype changes.
- Apoptosis is a common endpoint assessed in miRNA functional studies.
- The choice of cell dissociation enzyme can potentially influence cellular viability and apoptosis measurements in subsequent assays.
Purpose of the Study:
- To investigate the differential impact of four common cell dissociation enzymes (TrypLE, Accutase, Trypsin, Accumax) on cell viability and apoptosis.
- To evaluate the influence of these enzymes on Annexin V flow cytometry results following miRNA-based transient transfection.
Main Methods:
- Epithelial cancer cells (UM-SCC-12) were transiently transfected with a nonsense oligo for 24 hours.
- Cells were subsequently harvested using TrypLE, Accutase, Accumax, or Trypsin.
- Annexin V/7-AAD assay was performed using flow cytometry to assess apoptosis and cell viability.
Main Results:
- Trypsin treatment resulted in significantly lower cell viability and apoptosis rates compared to TrypLE and Accutase.
- miRNA transfection influenced cell viability and necrosis, particularly in trypsinized cells, affecting Annexin V assay outcomes.
- TrypLE exhibited the lowest inter-assay variability (1.13%), indicating superior reproducibility and reliability for this cell line.
Conclusions:
- Cell dissociation enzymes have variable effects on transfected cells, potentially introducing errors in apoptosis detection via Annexin V assay.
- Pre-assay testing of cell dissociation enzymes on transfected cells is recommended to ensure accurate and reproducible miRNA functional analysis.
- Standardizing enzyme selection is critical for reliable miRNA research.
