Tuesday, August 28, 2018

A Matter of Time

When evaluating treatment-induced effects on cells, timing is critical. Cells respond to changes in their environment in diverse ways – from sub-second signaling cascades to changes in cell health that accumulate over days to weeks. The ability to characterize events occurring over these markedly different timescales provides unique insight into cellular processes and increased flexibility for drug development studies.

End-point assays generate a single snapshot of these processes – often at an arbitrarily chosen time point – that is incomplete and easy to misinterpret. In contrast, kinetic imaging-based assays provide a detailed profile of cell characteristics over time, enabling unique quantitative analysis and intuitive validation of results.

BioTek Instrument’s versatile automated imaging systems allow researchers to monitor live cells over a full range of time courses, from seconds to weeks. The instruments maintain optimal environmental growth conditions for long-term studies of cell stress and viability, while an image capture rate of up to 20 frames per second, and aligned dual reagent injectors, enable characterization of rapid cell signaling events.

An example of the unique experiments that are possible with BioTek’s Cytation™ or Lionheart™ FX imaging systems comes from a recent study using expressed biosensors from Montana Molecular. Chemically-induced cell stress by thapsigargin, a potent SERCA pump inhibitor, was measured over a 24 hour period using a new cell stress sensor, while effects on Gq-mediated cell signaling where simultaneously monitored with the R-GECO calcium sensor.

Monitoring stress over 24 hour period in HEK293 cells treated with 1 µM thapsigargin.

These results reveal that cell stress induced by thapsigargin is detected at an order of magnitude lower concentration compared to discernible effects on cellular proliferation. Stress levels peak after 6 hours of treatment. After which, cells either recover naturally from this stress within 22 hours (e.g. 0.1 and 0.3 µM thapsigargin) or progress to more advanced states of distress (i.e. mitotic arrest or cell death).

The effect of thapsigargin on Gq-dependent Ca2+ signaling was probed by adding hM1 receptor agonist to treated cells using the reagent injectors. Ca2+ signaling was reduced in a dose-dependent manner at both 6 and 22 hours during thapsigargin treatment, indicating that the effects of ER stress on Gq-mediated Ca2+ signaling remain even after the cell has shut down the stress response.

Monitoring Gq-dependent calcium signaling in HEK293 cells 6 hours after 1 nM thapsigargin treatment

Click on the image to see larger version.

The ability to monitor cell stress and associated effects on cell signaling is important for understanding a broad range of diseases, as well as for the process of developing drugs designed to treat them. The unique combination of kinetic assays that are possible with BioTek automated imaging systems provides researchers with more physiologically relevant insight and expands research possibilities.

Visit our website to learn more about the full range of application solutions available with BioTek Instruments.

By: BioTek Instruments, Joe Clayton, PhD., Principal Scientist

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