Induction of Apoptosis Using an Active Microfluidic System for Microscopy
Poster presented at Lab Automation 2008
D. Ling, L. Higashi, A. Cabasug, S. Henderson, D. Poon
Apoptosis is a programmed physiological mode of cell death that plays an important role in tissue homeostasis. Understanding the basic mechanisms that underlie apoptosis will point to potentially new targets of therapeutic treatment of diseases that show an imbalance between cell proliferation and cell loss.
Conventional methods used to record cellular response in drug-induced apoptosis assays typically require dedicated personnel for the entire time-course, frequent disruption for microscopic observation, and potentially high variability. Nanopoint's live cell imaging and microfluidics platform enables precise monitoring and recording of live cellular processes and responses to external stimuli such as drugs, environmental changes, and growth factors. Benefits also include automated repeatability, reduction of materials and reagents used, multiplexed experimentation, and statistically significant data results.
An assay was used to distinguish between healthy and apoptotic cells based on mitochondrial membrane potential disruption. In non-apoptotic cells, JC-1 stain accumulates as aggregates in the mitochondria, resulting in red fluorescence. Conversely, in apoptotic and necrotic cells, JC-1 exists in the monomeric form and stains cells green. To induce apoptosis, cells were dosed with the potassium-specific transporter Valinomycin. A time-lapse experiment is described using the cellTRAY Live Cell Imaging System that effectively captures short and long term maintenance of healthy cells and the process of healthy HEK293TRex cells undergoing apoptosis following drug dosing. Well-to-well consistency is demonstrated by near-simultaneous cellular response to the drug within the entire region.
Region-to-region isolation and well-to-well consistency across an entire region
4µM Valinomycin-induced apoptosis
HEK293 JC1 mean intensity vs. time