Expert advice: Navigating reagent dispensing for MTT assays
Gerald Posch, a biotechnologist and Product Manager with INTEGRA Biosciences, specializes in optimizing the often overlooked but significant details that affect experimental success. Gerald earned his master’s degree and his PhD from the University of Natural Resources and Life Sciences, Vienna, where he investigated enzyme kinetics, characterized bacterial cell surface properties, and studied bacterial glycosylation, the subtle post-translational addition of sugar moieties to proteins that affect protein dynamics. Gerald applied his expertise in glycosylation engineering to vaccine development during his postdoctoral research at the University of Alberta and in the biopharmaceutical industry for 5 years, before transitioning to INTEGRA. Using his eye for detail and talking to researchers on the forefront of drug discovery, Gerald and the INTEGRA team developed the WELLJET reagent dispenser as an innovative solution to help ease the tedious pain points of reagent dispensing for common assays, such as the MTT assay.
How does using a reagent dispenser improve MTT assay data?
Dispensing cells and the different solutions that make up an MTT assay needs to be very consistent, which might not be the case if one sets up this assay by hand using simple pipetting. Having an automated dispensing solution helps data reproducibility and consistency. The WELLJET uses a peristaltic pump system that propels the liquid forward via a rotor integrated into the pump unit of the dispenser. Unlike other reagent dispensers, the WELLJET uses the EasySnap™ cassette, which is made from injection-molded silicone, to ensure that every tube in the cassette has the same dimensions. The EasySnap cassette design, coupled with the peristaltic pump, provides consistent reagent dispensing across cassette channels, meaning that researchers can rest assured that the same correct volume is applied to each well of their multiwell plate. This eliminates well-to-well variability and safeguards experimental accuracy. The liquid within the silicone tubing doesn’t interact with the instrument or pump parts, protecting experimental sterility. The WELLJET can aspirate liquids from different source vessels, which can be anything from bottles to flasks to tubes, and then dispenses the liquid across any size multiwell plate from a 6 well to a 1536 well plate. This allows researchers to tailor their reagent dispenser to meet the unique needs of different experiments and experimental steps.
Changes in cell viability could affect MTT assay results. How can using a reagent dispenser help optimize cell viability?
The main advantage is that it’s a very gentle process. The cells are pumped through the silicone tubing at very low shear forces, which is ideal for fragile cell lines. We have 2 types of dispensing cassettes: a large bore dispensing cassette, and a small bore dispensing cassette. The large bore dispensing cassette not only has a wider bore size for the silicone tubing, but also a larger orifice for the dispensing nozzle. If someone is not yet experienced with reagent dispensing because they’ve always used pipettes, and they want to start dispensing cells, then the first recommendation we give is to use the large bore dispensing cassette with the wider orifice, just to make sure their cells will be happy, especially if the sensitivity and fragility of the cells is unknown. But we have data from customers who have successfully used the small bore cassette with fragile cell lines and at high dispensing speeds.
What other factors should researchers consider when dispensing cells?
It really depends on the cell type for troubleshooting. In general, dispensing speed is important. We have 3 different speed settings for the WELLJET: fast, medium, and slow. We have seen good results with the fast speed setting. If one sees that the cells are not as viable, then slower speeds should be used for further optimization. Also, for other troubleshooting methods, the height of the dispensing module above the plate can have an influence. Finding the right height setting or lowering the dispense height may help researchers get better results for fragile cells.
How should researchers care for their dispensing cassettes to maximize cassette lifespan?
The cassettes are reusable. The standard lifetime in terms of plates that can be dispensed is 2000 96 well plates at 100 microliters. After that, it’s recommended to change the cassette. INTEGRA supplies sterile cassettes but, to maintain cassettes in optimal condition, it is important to properly clean them after each dispensing campaign. Dispensed cell solutions, buffers, or compounds contain cells, proteins, and salts, which may cause the cassette to clog, especially at the point of the dispensing nozzles. We recommend that scientists thoroughly wash the cell solution out with deionized water and a mild detergent. They then need to wash out the detergent with water to avoid residual cleaner sitting in the tubing or the cassette when starting the next dispensing campaign with cells. After cleaning, it is recommended to rinse the cassette with a disinfectant. Scientists can set up a cleaning program for these steps, and they need to move the tubing into different source vessels. So, it’s not a fully automated process, but we wanted to give customers guidance by allowing them to define cleaning programs for the cassettes. Alternatively, the cassettes can also be autoclaved to ensure sterility.
How can scientists use reagent dispensing in automated workflows?
The dispensing process is automated and, with the dispenser stacker version, scientists can process a whole stack of plates to dispense cells or reagents. In that sense, these instruments are standalone units. However, the WELLJET can also integrate into automated workflows using application programming interface (API) commands. We provide the instrument and API commands that allow an integration company to speak with our system. This triggers directions that allow the instrument to be used in a fully automated system. There are integration companies that combine instruments from different providers to work in concert. For instance, for a full cell-based assay, the WELLJET only dispenses cells or reagents, but a scientist might also want to have an optical readout or incubation steps in between reagent dispensing. This can be handled by a robotic arm and a master software, provided by an integration company. The robotic arm shifts multiwell plates from one instrument to the other, and starts the processes on the instruments. In the end, you can automate a whole assay or a whole drug compound screening platform.
In what other applications can researchers use a reagent dispenser?
Almost every assay requires some sort of reagent dispensing or liquid handling. As such, reagent dispensers can help with almost any experiment. The WELLJET serves plates that meet ANSI/SLAS format, the standard format defined for multiwell plates in terms of plate dimensions and how the different wells are set up. Scientists can start with a single plate and go up to multiple plates, and then, if they need higher throughput, they can switch to the stacker version. Both instruments are designed to have very small footprints to fit inside a biosafety workbench.
What is the most innovative feature of the WELLJET reagent dispenser?
The most innovative feature for me is the EasySnap dispensing cassette, which uses molded silicone tubing to avoid recalibration, cutting the cost for our customers and ensuring high accuracy and precision across all channels. Also, the large, easy-to-navigate interface combined with very smooth and precise mechanics, sets the WELLJET apart from other reagent dispensers. We put as much technology as possible into a very compact instrument. That’s always the challenge, to make an instrument as compact as possible without compromising on its functionality. We go out and speak with scientists to understand their pain points with existing solutions, and how we can improve those areas with new solutions. We are not aiming to just make another product. We want to make sure that what we develop is also at the forefront of innovation. In the end, it’s very exciting to talk with customers and hear how it’s helping them in terms of efficiency, data generation, reliability, and consistency.
If you would like to get additional information on this topic, please download our technology guide for applying reagent dispensing to MTT assays:
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