Induction of Apoptosis Using an Active Microfluidic System for Microscopy

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.

Experimental Procedure

1. CT-2000 cellTRAYs were seeded with HEK293TRex cells at a concentration of 1.0 x 106 cells/mL one day prior to experimentation.
2. Stock JC1 stain (Cell Technologies, Inc) was diluted 50-fold with 1x JC1 assay buffer. A 4mM and 1mM Valinomycin working solution was prepared in 1x JC1 assay buffer.
3. Growth media was aspirated and 1mL of diluted JC1 stain was added directly onto the cells. Cells were placed back into the 37°C incubator for 30 minutes.
4. Fluidic assemblies were flushed and primed with 0.22mm filtered growth media.
5. Stain was aspirated and the cells were washed 3 times with 1mL of 1x assay buffer.
6. The CT-2000 cellTRAY was seated onto the microscope and attached with the cellTRAY Manifold.
7. Microfluidics, temperature, and CO₂ were started.
8. Baseline brightfield and fluorescent (red=alive and green=dead) images raster scans were captured to document the condition of the cells.
9. Microfluidics were stopped and 10µL of 1x assay buffer or valinomycin (4mM or 1mM) were infused in “fast feed mode” for 2 minutes.
10. Changed growth media feed to 20 seconds pulse delay setting; T=0 fluorescent images were captured.
11. Maintained 20 second pulse delay setting for 12 minutes; changed to “normal flow” setting. Start automated time lapse fluorescent image scans.
12. Process images using Nanopoint's Scan Reorganizer utility and ImageJ.

Results

Region-to-region isolation and well-to-well consistency across an entire region

4µM Valinomycin-induced apoptosis
HEK293 JC1 mean intensity vs. time

No Valinomycin

1µM Valinomycin