TY - JOUR T1 - Cell Dissociation Enzymes Affect Annexin V/Flow-Cytometric Apoptotic Assay Outcomes After miRNA-based Transient Transfection JF - Anticancer Research JO - Anticancer Res SP - 2819 LP - 2825 DO - 10.21873/anticanres.15763 VL - 42 IS - 6 AU - CHRISTINA M. GOBIN AU - JAMES N. MENEFEE AU - CHAYIL C. LATTIMORE AU - ANDRIA DOTY AU - KRISTIANNA M. FREDENBURG Y1 - 2022/06/01 UR - http://ar.iiarjournals.org/content/42/6/2819.abstract N2 - 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. ER -