Tips for Designing Effective Workflows in a Microplate Environment

Principles of effective workflow design are universal to any application. Good workflow design and versatile instrumentation are critical components for making good investment decisions in meeting throughput requirements for any laboratory utilizing microplates. The EL406 Combination Washer\Dispenser offers versatile workflow options as it provides multiple microplate washing and dispense options in a single instrument. A few important workflow advantages of the EL406 are decreased downtime, as there is less instrumentation requiring routine maintenance; increased throughput, as there are less robotic steps required during assay processing and all fluidics can be supplied and resupplied at only one station; and, cost effectiveness, as there are few if any consumables required for washing and reagent dispensing to the microplate and the small footprint of this one multi-use instrument saves bench space. Here are some tips we have compiled for designing effective microplate-based workflows that are practical for a variety of applications and throughput needs.

1. Look for potential bottlenecks in workflow by understanding throughput limitations of the assay(s) (e.g. incubation times or reagent volumes) and the instrumentation (e.g. dead volumes and prime volumes), and then design workflow around these first.
2. Perform simulated throughput models before ‘going live’ with an assay workflow.
3. Treat validation of the scheduling as importantly as validating assay performance on an instrument, and take time to test different scenarios.
4. Start with the potentially highest throughput for a testing facility and map how to get there within the given labor and budgetary resources available. This will help identify areas that are already strong, even if they are manually performed, and areas that are weak and may benefit from automation. Consider automation that can reflect currently successful workflows.
5. Optimize assay performance on the instrumentation before designing a final workflow. The process of confirming ideal assay performance often results in a specialized combination of tolerances that ultimately impact final workflow. For example, if an assay performs best when a 2 minute soak is integrated into the wash step, it is best to know that before designing the final scheduling scenario.
6. Plan on some assay waste to validate the model and rework it if necessary.
7. Build up to a final throughput if possible, adding additional throughput capacity after a single workflow has repeatedly worked effectively.
8. Have a backup workflow that can be run with minimum instrumentation if major components go down for any reason.
9. Invest in automation that is adaptable, easy to use, easy to edit, and easy to upgrade either through a single source manufacturer, or a proven group of collaborative components. There are sophisticated scheduling software products available, but they may be challenging to program, or have limited compatibility with a broad range of instrument manufacturers. Support options for these products may make it difficult to change or add throughput scenarios to the original model.
10. Workflow changes ‘on the fly’ can consume time and material costs that can be alleviated by planning the right combination of compatible instrumentation before purchasing – even if they are not from the same source. Try to plan for what can go wrong by targeting what the strengths and weaknesses of each component in the workflow are and make investment decisions based on the best balance between each piece of automation.

We would be interested in learning any tips you may have.

Governor of Vermont Opens BioTek’s Applications Lab

On Friday, Dec 11, Governor Jim Douglas of Vermont visited BioTek and officially opened the new Applications Lab. Governor Douglas was given a tour of the lab which highlighted its capabilities for performing cell-based assays using BioTek instrumentation.

Scientific Director, Peter Banks demonstrates the Epoch Multi-Volume Spectrophotometer System to Governor Jim Douglas

The new Lab is double the size at over 1,100 sq. ft. compared to the previous Applications Lab, but the most important addition is a dedicated cell culture room of 325 sq. ft. Within that space, we have:

• 16' of laminar flow hood space to perform tissue culture manipulations, including passing and plating cells.


• Three of the hoods will be used for eukaryotic cell manipulations; one will be used for bacterial cell processing.


• Each of the hoods is large enough that we can easily fit BioTek's liquid handling equipment inside the hood. This makes for easy robotic manipulation of sterile cells and media in an aseptic environment.


• Four incubators combined provide us with 19.5 cubic feet of space to grow eukaryotic cell lines.


• Because we possess multiple incubators, cell lines can be grown that require varied temperature or CO2 conditions.


• The incubators are large enough that we can fit new culturing instrumentation inside the incubator, which will allow us to generate project data using next generation culturing techniques.

With this new facility we will broaden and expand our collaborations with reagent partner companies, customers and the scientific community in general.

Synergy H4 Hybrid plate reader. What? Why?

I purchased a Dodge Durango a few months ago, and as a family car, I love it. There is plenty of room to carry people and things around, it has all sort of gadgets (the DVD player for the kids being #1 on my list of favorites) and we all feel safe riding high above the street. But I have to say that the 5.7 L V8 Hemi engine is not exactly what I would call a green and economical mechanical system. When we chose this car as our family car, the emphasis for us was on interior space, safety, and kid’s friendly gimmicks (and being from Europe, also a touch of “I want a big American car at least once in my lifetime”!). Now, I wish the car had a button that would turn it into a Prius whenever I just need to drive to the grocery store, but no such car exist. Don’t you hate compromise? And more importantly, what does all this have to do with plate readers?

Cars are what I often have in mind when we, at BioTek, work on developing new multi-mode plate readers. Like cars, multi-mode plate readers come with all sorts of options and accessories, and are available in a broad price range. Like cars, they have become an object without which many activities would become impossible, or extremely cumbersome. And like cars, they are available from a variety of vendors that fight for the customer’s attention. And so when we develop a new plate reader at BioTek, I often have in mind my own purchasing experience with cars: how did a specific model or vendor get my attention? How did the test drive feel? And how do I feel today, after a few years of driving?

I believe BioTek’s new Synergy H4 is a great example of how we try answering such questions, as a scientific instrumentation company. Synergy H4 is a Hybrid multi-mode microplate reader, “Hybrid” because you can choose to use filters or monochromators for wavelength selection. As I said above, there is no car that you can turn from a gas-guzzling SUV to a small fuel-efficient hybrid with the push of a button. But there is now a plate reader that will switch from deep blocking interference filters to quad monochromators with a click of your mouse. Why is this meaningful? Because microplates are just vessels. What scientists put in these vessels is only limited by their imagination (and budget!). Ask our technical assistance staff what some of our customers try to do with their plate readers! If plate readers were alive, I can guarantee there would be a plate reader rights movement! The range of applications is just mind-boggling. This is where our hybrid design comes to play: when we (BioTek) create instruments that push the limits of flexibility, we open new horizons to researchers. The tool becomes less visible, gets out of the way of the research process and leaves room for more creativity and productivity. The “Hybrid” concept can catch people’s attention because of its uniqueness, but beyond that, it delivers a combination of flexibility and performance which feels like driving a race car AND a Jeep at the same time.

When launch time came closer, we needed a name for our filter/monochromator combination. In the end, after much brainstorming, we went with "Hybrid Technology^TM". And even though the ^“TM” might stand for “Totally Marketing” (WE WANT YOUR ATTENTION, remember?), the actual hardware and software are just incredible. Don’t just take my word for it. Try one. We have demo instruments, friendly and knowledgeable sales and support staff around the globe. And once you have one in your lab, you will be among the growing numbers of scientists that just wonder: why did I wait so long before getting a hybrid plate reader?

Fluorometric Quantitation of dsDNA using Picogreen

Double stranded DNA (dsDNA) is commonly quantified using spectrophotometry at 260 nm without need of additional reagents. However, quantification is limited to 50 ng/mL of dsDNA (0.001 OD) and more typically between 2 and 200 ug/mL in a microplate well sample that is pathlength corrected to 1 cm. Other substances present in dsDNA samples can also absorb at 260 nm including nucleotides, ssDNA, RNA, EDTA, and phenol and skew results ⁽¹⁾. Quantification below these concentrations typically reaquires the addition of reagents, such as fluorophores with selectivity for dsDNA.

Fluorescent dye Hoechst 33258 is inexpensive and more sensitive that spectophotometry allowing quantification down to 3 ng/mL of dsDNA. Quantitation requires a standard curve and for the best results, top reading solid black plates is recommended. It can bind to ssDNA, however ^(1,2).

Picogreen® is also a very simple test consisting of TE buffer, Picogreen reagent, a standard curve formed by supplied dsDNA standards and customer supplied samples. Because Picogreen is extremely sensitive, the 20x TE buffer included in the Quant-iT™ PicoGreen dsDNA Assay Kit is certified to be nucleic acid–free and DNase free. The 1x TE working solution is prepared by diluting the supplied 20x buffer with sterile, distilled, DNase free water. Life Technologies (Invitrogen) advertises a dynamic range from 25 pg/mL to 1000 ng/mL ⁽²⁾. Our application note Fluorometric dsDNA Quantification Using PicoGreen® exhibits linearity over the entire dynamic range ⁽³⁾. Average CV's with the Synergy Mx was 2.6% over a 12 standard dynamic range from 0 to 6.4 ng / mL ⁽⁴⁾.

Some Tips For Using PicoGreen

• Standard curve dilutions should be prepared in plastic and not glass tubes to avoid the reagent sticking to the glass
• Picogreen reagent is sensitive to light and wrapping the tube containing the working reagent with tin foil is advisable
• Picogreen reagent is stable for 3 hours and prepared plates, if covered to reduce evaporation and kept from light, for a least an hour more
• Aliquots from the tubes are pipetted into the microplate with a total volume of 200 uL per well. Preparing the standards directly in the microplate worsens CV's
• Samples are best prepared in tubes with the TE buffer but can be prepared in the microplate by adding the Picogreen® working reagent to the wells followed by the sample
• Detection limits depend on quality of DNA, buffer source, age and storage conditions of Picogreen reagent and the optical quality of the reader and wavelength selections
• High salts such as 200 nM sodlum chloride and 50 nM of magnesium chloride can depress the signal
• Because of the extreme sensitivity of this assay, gloves should be worn at all times even when removing microplates from the source box for the plates.
• Solid black plates such as Corning 3915 provide the lowest background although a few customers report better results for their assays with Corning 3615 low background clear bottomed plates using bottom reading
• Because of the large dynamic range of the assay, photomultiplier sensitivity setting (gain) should be set high enough so that lower concentrations can be easily differentiated from the blank

References

(1) Held, Paul (2009), [Scientifically Speaking] Quantitation of dsDNA using Hoechst dye 33258, BioTek Instruments, Inc website blog, Winooski, VT

(2) Quant-iT PicoGreen dsDNA Assay kit instruction manual MP07581 "Quant-iT PicoGreen Reagent and Kits".

(3) Held, Paul (2006) Fluorometric dsDNA Quantification Using PicoGreen®, BioTek Instruments, Inc, Winooski, VT

(4) Quigley, Ted (2009), Experimental data