Thursday, February 20, 2014

Why Choose Cytation™ 3: Improving Data Generation Efficiency and Understanding from Cell-Based Assays


A large percentage of cell-based assays incorporate fluorescence or luminescence detection that are easily quantified using conventional microplate readers, even in higher throughputs. Quantitative data is rapidly generated, allowing users to make quick decisions; however, this may not always tell the entire story about what’s happening with the cells following treatment. This can lead to false assumptions and the wrong path forward in subsequent experimentation. In contrast, cell-based imaging assays allow visualization of events at the cellular level. However, image capture and storage can require considerable time and computer or network storage space.

This leads us to two reasons why someone would choose our Cytation™ 3 Cell Imaging Multi-Mode Reader instead of a separate imager and microplate reader when performing cell-based assays: confirming cellular activity and hit-picking. We performed two experiments to illustrate these benefits.

Cellular Imaging Confirmation

To illustrate the importance of confirming cellular activity, we repeatedly dosed hepatocytes with a drug over multiple days and assessed cell viability/toxicity. This is very similar to hepatotoxicity studies performed in the drug discovery process following lead molecule generation. Cryopreserved human hepatocytes (BioreclamationIVT, Baltimore, MD) were cultured in 96-well collagen-coated plates at a concentration of 50,000 cells/well. Several concentrations of the known cytotoxin, camptothecin, were added to the cultures on a daily basis over a seven-day period. Potential cytotoxicity from the compound was measured at 1, 3, and 7 days following treatment initiation. Total and cytotoxic cell numbers were assessed using the Hoechst 33342 cell permeable nuclear stain, and CellTox™ Green Cytotoxicity Assay from Promega Corporation (Madison, WI), respectively. All measurements were performed using Cytation 3.

By looking at results from the mean green fluorescence using the CellTox Green dye (Figure 1), it is apparent that fluorescent values increase in a dose- and time-dependent fashion for days 1 and 3, indicating that the cytotoxic effect from the compound is also increasing in the same manner. However on day 7, cytotoxicity increases rapidly at lower camptothecin doses, but then falls off to levels below the uninhibited control at higher concentrations. If one only had access to this data set generated by a microplate reader, it would be difficult to assess exactly what effect the higher camptothecin doses have on hepatocytes after a seven-day dosing. However, as we show next, by imaging the well, it then becomes clear as to why the fluorescence is “disappearing” from these wells.

The captured 4x images of hepatocytes treated with 20,000 nM camptothecin (Figure 2) illustrate how cytotoxicity increases from day 1 to day 3. Total cells show up as blue, while dead cells are seen as green. After a seven day dosing, though, the camptothecin concentration is completely toxic to the cells, causing them to detach from the bottom of the well and float up into the media. Only by incorporating imaging would this phenomenon be noticed, allowing for the correct conclusion to be drawn about this compound.


Figure 1.  Hepatotoxicity results following 1, 3, and 7 day camptothecin treatments.
Figure 1.  Hepatotoxicity results following 1, 3, and 7 day camptothecin treatments. 


Figure 2. 4x images of hepatocytes treated with 20 µM camptothecin showing live (blue) and dead (green) cells
Figure 2. 4x images of hepatocytes treated with 20 µM camptothecin showing live (blue) and dead (green) cells.


Hit Picking

In high content phenotypic assays, every microplate well is imaged, even though only a few wells may ultimately contain data of interest. This takes a great deal of time, and requires lots of data storage. Cytation 3 may be programmed with an intensity threshold that triggers the instrument to image only those wells that meet, or “hit”, a user-programmed criterion when the plate is read instead of the entire plate. This hit picking dramatically reduces analysis time and data storage requirements for greater efficiencies.

To illustrate the “hit picking” process, we performed an experiment to identify potential cellular hypoxia inhibitors. Immortalized keratinocytes were cultured in the presence of cobalt chloride (CoCl2), a known inducer of hypoxia-like responses, along with a REDOX compound library (Enzo Life Sciences, Farmingdale, NY) made up of several known antioxidants. Hoechst 33342 cell permeable nuclear stain and fluorescence-based Cyto-ID® Hypoxia/Oxidative Stress Detection Kit from Enzo Life Sciences were used to identify cell numbers and assess inhibition of oxidative stress and hypoxia. Prior to reading the assayed microplates, a single Cytation 3 Hit Pick protocol was created. The protocol used the results from a monochromator-based read of the signal from the hypoxia dye to initiate imaging of wells whose RFU values were greater than one standard deviation lower than the average of four positive control wells. This corresponded to greater than or equal to 50% inhibition of the hypoxic condition.

Per Figure 3, a total of 27 wells were imaged using the protocol, including 15 "hit picked" wells and 12 control wells in row H. The time to capture three images from these wells is 3 minutes and 22 seconds. When compared to the total time required to capture three images from all 96 wells, which is 12 minutes, this represents a 3.5 fold savings in time, as well as required storage space.


Figure 3.  Selected wells for imaging using “Hit Pick” protocol criteria.
Figure 3.  Selected wells for imaging using “Hit Pick” protocol criteria.

Summary

Cell-based microplate reader and imaging-based assays play an essential role in current life science research. While both yield important information necessary for making critical decisions, each can suffer from crucial shortfalls when performed on an instrument only capable of performing one type of detection.  The Cytation 3’s combined capabilities, however, alleviate these concerns by providing an instrument which can streamline processes and ensure that all relevant experimental data is available during the decision making process.


Want to learn more? Check out these pages:

By: BioTek Instruments, Brad Larson, Principal Scientist

No comments:

Post a Comment