Our support for researchers extends beyond our team of scientists at the product development level. We have teams of field application scientists (FAS) based around the world that are ready to support your research at any level, whether that be by advising on experimental design or by helping you secure custom and bulk orders of the reagents you need. We interviewed a few of them to get their thoughts on where the field of flow cytometry is headed.

 

Once you’ve read through their thoughts, you can reach out to them for additional help.

 

 

Our field application scientists have decades of combined experience helping researchers with their flow cytometry questions. From left to right, pictured above are Leesa Pennell (LP), PhD, Lead Technical Application Scientist, based in the US; Samuel Kӧrner (SK), PhD, Technical Application Scientist II, based in Germany; and Hongyan An (HA), PhD, Technical Application Scientist I, based in Australia.

 

How does your expertise help customers have successful flow cytometry experiments?

 

LP: As an immunologist who has been around flow cytometry for over 10 years and worked at BioLegend for over five, my personal experience can help with our clones, fluors, and building panels on different instrumentation. I have done varying types of experiments (in vitro, ex vivo, human, mouse, cytokine assays, etc.), all with their own nuances that are important to keep in mind for a successful experiment. The feedback I get from building panels for customers helps me tailor my approach to each future panel I work on. I then use each of these tools for my customers to ensure they’re getting support with their experiment, from start to finish. While a well-thought-out panel makes things go a lot smoother, I wouldn’t say it’s the be-all and end-all! Researchers need to do their part in making sure to test out the panel in their own experiments, have all the relevant controls, etc. For that reason, I feel my part is the easy part!

 

SK: My academic background allows me to understand the challenges many scientists face while working on their specific projects. One of the interesting aspects of being a field application scientist is that you get to combine your own hands-on experience with customer feedback to support researchers in the best possible way with their flow cytometry experiments.

 

HA: I built up my flow knowledge through my PhD, post-doctoral training, and time as a BioLegend FAS. The latter has me working closely with various researchers, helping them design flow panels for different cell types. FASes have the most up-to-date knowledge of flow reagents, such as new fluorophores, buffers, and unique antibody clones. Most importantly, we are committed to fully supporting researchers by providing technical assistance and solutions for panel design, optimization, and expansion.

 

What specific tips would you provide to ensure that you have a multicolor panel that is successful?

 

LP: Theory will only get you so far. Also, what works for someone, may not work in your own hands. Every panel should be tested rigorously for performance and resolution. Accurate and adequate controls are very important too. An experiment can only be as good as its controls. Work with your flow cytometry core, our web tools for flow, BioLegend FASes, and colleagues to ensure all of the correct controls are done and the instrument is performing as expected. Don’t bite off more than you can chew - think about what you want to get from the data, not just the capabilities of your cytometer. Just because it’s possible to do a 40+ parameter experiment doesn’t mean it’s necessary or compatible with your hypothesis and desired targets/experimental outcome.

 

SK: When helping with panel building requests, one needs to take into consideration specific requirements like different flow cytometer settings, cell types, gating strategies, antigen expression, etc. One piece of advice would be to use specific scientific resources, e.g., the Optimized Multicolor Immunofluorescence Panels (OMIP). If possible, I would then recommend building a multicolor panel in phases. Once your initial panel is working, you can implement more markers to expand it further. The more complex a flow experiment gets, the more optimization it might require after the initial panel was designed, e.g., proper antibody titration, correct setting of voltages, and the usage of appropriate controls. If have any doubts about your panel, feel free to reach out to your local representative. We are always happy to help.

 

HA: Understanding and managing fluorescent spillover is critical for building a multicolor panel and attaining high-quality data. When fluorophores spill into the detectors of other fluorophores, it can cause significant background and affect the accuracy of the signal. Compensation or spectral unmixing can correct this for conventional or spectral flow cytometry, respectively. Additionally, it can help to titrate antibodies and assign mutually exclusive makers for dyes that spread significantly into each other. You can find more on our webpage regarding best practices, sample preparation, and controls.

 

What are some common mistakes and misconceptions you often see and hear related to flow cytometry experiments?

 

LP: A lot of times, it’s the user’s controls. Many users try to skirt around doing proper controls, and it will come back to affect them at some point, so I really try to emphasize this when working with a new customer. Many users don’t titrate antibodies to make sure their staining is optimized for their application, which is so important! Finally, instrument setup is often done poorly or incorrectly, which can lead to acquisition of misleading or incorrect data. Flow cytometry cores can be invaluable for obtaining proper training from cytometrists. Unfortunately, bad habits can snowball, so it’s best to try and access a flow core or training course before proceeding with complex panels.

 

HA: Some researchers assume that seeing a fluorescence signal in the donor channel of a tandem dye is degradation. It is important to note that tandem dyes are never 100% efficient at transferring energy (a process known as FRET), meaning that some spillover or bleeding in the donor channel is expected. Energy transfer efficiency depends on the acceptor-to-donor molar ratio, distance, and orientation. Suppose you observe a strong signal in the donor channel and a weak signal in the acceptor channel. This does not necessarily mean that the donor and acceptor molecules fell apart. It just indicates that FRET efficiency is low, possibly due to photobleaching, exposure to freezing temperature or fixatives, and permeabilization.

 

Is there a memorable panel you built for researchers?

 

SK: Yes, a 29-color panel for the analysis of human T cells. The panel included five chemokine receptor and six transcription factors, making it challenging to design. A couple of follow-up meetings were necessary in order to review the initial data and optimize the panel and staining protocol.

 

HA: Understandably, researchers want to maximize the number of markers in their panel because it can provide a comprehensive analysis and save precious clinical samples. However, it may impact the data quality due to the instrumental capacity. One time, a researcher wanted to assess over 25 intracellular and surface markers simultaneously with a three-laser Aurora instrument. After discussion, I helped build two 22 parameter panels instead, aiming to study human T cells and B cells separately for better data resolution.

 

Where do you see the future of flow cytometry headed?

 

LP: We’re now into the 40+ parameter space in full spectrum flow, so I expect panels will continue expanding as new fluors are developed to fill the entire spectrum. I think it’s really neat to be part of a company that is helping to fill those gaps for researchers and really flesh out their panels so they can get the most information possible from a small sample. I wish I had the ability to do 30 parameter experiments when I was in grad school so I wouldn’t have had to run three panels on all my samples.

 

HA: I anticipate that future of flow cytometry will provide a much broader and deeper analysis, allowing more surface and intracellular markers to be analyzed simultaneously. Advancements in technologies, including advanced instrumentation, new fluorophore development, the discovery of new antibody-binding epitopes, and automated analysis software are the key to driving this adoption in the coming years.