Cytotoxicity

Systematic automated phenotypic screening of living cells; profile cell responses to drug-perturbation(s) in parallel, to accelerate drug discovery in biotech and pharma
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Dose-response curves provide important pharmacodynamic properties (efficacy, potency, toxicity, and lethality) for evaluating the performance of candidate drugs in the discovery pipeline. Half maximal effective concentration (EC50) values are a standard indicator of drug potency, but the robustness and reproducibility of EC50 values generated using current methods is low.

The non-invasive nature of label-free imaging means cells can be continuously monitored, over infinite periods of time, which allows kinetic EC50 values to be calculated. Kinetic EC50 values provide information about a drug’s stability; whether a drug’s potency increases or decreases over time.

Label-free timelapse images showing dose-dependent cell death in HeLa cells following exposure to Chloroquine over 60 h

Dose-dependent cell death in HeLa cells following exposure to Chloroquine over 60 h

In this experiment, HeLa cells were grown in standard culture conditions and plated in 25 wells of a fibronectin coated 96-well plate. One row was kept as an experimental control and Chloroquine was added at 4 concentrations to the other 4 rows. Images were captured using Nanolive’s automated microscope, the CX-A. One image was taken every 15 minutes until death was observed in all Chloroquine treatments, a period that took 60 h.

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Changes in the total dry mass of HeLa cells following exposure to Chloroquine

Mean total dry mass graph Green
Mean total dry mass graph Yellow
Mean total dry mass graph Blue
Mean total dry mass graph Brown
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Changes in the total dry mass of HeLa cells following exposure to control conditions (green), 25 µM (yellow), 50 µM (blue), 100 µM (brown), and 200 µM Chloroquine (red). Graphs show mean ± standard error (n = 5).

Using Nanolive’s AI-assisted proprietary software, EVE Analytics, images were analyzed and metrics extracted. Changes in total dry mass are an excellent indicator of cell death, so this metric was used to investigate cell death dynamics quantitatively and for the subsequent calculation of EC50 values.

No cell death was observed in control conditions and total dry mass increased by 44 % over the course of the experiment. Cell death was observed from the start of the experiment in cells exposed to the highest concentration of Chloroquine (200 µM), as is evidenced by a 68 % reduction in dry mass. After 40 h, all cells were dead.

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Calculating kinetic EC50 values

EVE Analytics outputs all metrics in .csv file format, allowing subsequent analyses to be completed in Excel.
There are 3 simple steps to calculating kinetic EC50 values:

  • Normalize data
  • Plot dose-response curves at single timepoints
  • Plot EC50 values over time

These steps are explained in detail in our application note below.

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Application Note

Feature Application: Calculating kinetic EC50 values from dose-response curves

Feature Application: How to calculate kinetic EC50 values

In this application note we show how to calculate kinetic EC50 values from data directly output from Nanolive’s image analysis software, EVE Analytics.

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Webinar

Watch our Webinar: How to calculate kinetic EC50 values with EVE Analytics

In this webinar, Dr. Emma Gibbin-Lameira shows how Nanolive’s label-free, non-invasive imaging can be used to quantify dose-dependent responses of HeLa cells to Chloroquine.

Specifically, Dr. Gibbin-Lameira shows that:

  • Dose-dependent experiments can be performed in 96-well plate format over long periods of time (> 2 days), reducing the need for time and labor-intensive multiple fixed-point measurements and eliminating cytotoxic/cytostatic effects arising from the use of labels, dyes, or phototoxic damage
  • Metrics output from Nanolive’s proprietary software, EVE Analytics, can be used to generate statistically robust time-dependent EC50 values based on total dry mass; a metric unique to label-free imaging
  • Sub-cellular spatial resolution combined with high content image analysis can be used to investigate a drugs’ mechanism of action

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