automated kinetic studies

There is a need for kinetic assays to be run on low-cost equipment. paa have teamed up with Molecluar device to produces a fully automated system for LaTrobe University in Melbource Australia.

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SYTOX Green stain is a simple and quantitative single-step dead-cell indicator for nucleic acid for measuring bacterial viability and its use is described elsewhere. Bacterial viability can be measured over a long time frame as a kinetic experiment, and with a fluorescent plate reader and 96-well microplates, a large number of experiments can be conducted at a time.  The problem is running the experiment over a time period of 24 hours.  If one plate is to run at a time, the plate can be left in the plate reader, however if multiple plates are to be run, the task becomes more a problem requiring lone time periods as one plate is run each day or shift work by the technical team.

This problem has been solved, and it is simple as 1-2-3 !

• Use a Molecular Devices SpectraMax plate reader and StakMax stacker
• Use Overlord3 from paa to control the process
• Use Microsoft Excel and a program written by paa to collate and display the data

SYTOX kinetic studies are time consuming and difficult to perform because of the time delays between plate reads, keeping track of the plates to read in the plate reader and collating the data.

paa in association with La Trobe University (Australia) and Molecular Devices have developed a low cost and reliable solution to this technique.

The hardware for this system consists of:

• Molecular Devices Spectromax plate reader
• Molecular Devices StakMax plate stacker (with optional barcode reader for plate tracking)

The software consists of:

• Overlord3 laboratory automation control software
• Microsoft Excel

Prepare the set of plates required for the kinetic assay, the Molecular Devices StakMax can handle 20 plates.

The requirement is to run this series of microplates in the plate reader once a day (or any other time delay). 

1. determine the time
2. get a plate from the input stack, read the barcode
3. open the plate reader plate drawer, load plate
4. close the plate reader drawer
5. run the required plate reader method
6. when finished open the plate drawer
7. get the plate from the plate reader
8. return the plate to the output stack
9. repeat steps 1-8 for all plates
10. restack the plates from output stack to in stack
11. wait the required time delay
12. run steps 1-11
13. run steps 1-12 until all the data has been collected

Overlord3 handles:

• Control of the instruments
• Running the process
• Timing the delay between read n and read n+1
• Collating the barcode data
• Passing the data to Microsoft Excel for data management and display

Overlord3 advantages:

• Ease of programming/Rapid assay development
• Increased plate throughput
• Reduced downtime - Fully recoverable from device errors
• Powerful design environment
• Support for device pooling

more information: Overlord3

The system records the data as the run proceeds and at the end of the run, the data is available for review.  One data file is produced for each plate in the run.

The system allows replicates to be placed on the plate.  A plate map in Excel allows the system to know where each sample is on the plate.  In the example (left), A1, A2 & A3 contain Sample 1, H10, H11 & H12 contain Sample 32.

These results are then averaged to give a result for each sample on the plate.  In the run above, each sample is summarised on a separate tab of the results workbook.

The advantage of producing the results in this format is the user can scan all the samples on the plate quickly and see the difference between a significant and non-significant result.  Compare the results below with that above.

 

The results for all the samples are displayed on a summary page at the front of the Excel workbook.

The use of Excel allows the user to then interpret the data further.

A simple pass/fail or significant result/non-significant result can be tagged to the data.

• the system capacity can be expanded with an automate.it scara robot
• the system can use a Thermo Cytomat or LiCONiC incubator to store the plates between reading at 37^oC or other temperature
• any time gap between reads can be stipulated
• any data handling can be incorporated as required

Using the data from La Trobe University, Australia, this technique of integrating reliable instrumentation from Molecular Devices with advanced scheduling software from paa, allows robust experiments to be planned with full plate tracking, data integration and data integrity.  With rapid data analysis, the time savings from the automation of this experiment are immense but the system is easy to use.

paa are grateful to Dr Nicole Van Der Weerden (La Trobe University) for sharing the results of her work and the example data shown above.

 

paa are grateful to Molecular Devices (UK) for the loan of the test equipment.