Ten Tips To Get You Started in Flow Cytometry

1 Ten Tips To Get You Started in Flow Cytometry Aja Rieger, PhD Flow cytometry is popular in cell biology and biomedical research,1,2 long viewed as a tool for immunologists.3 However, the real strength of this technology is that it can be applied to a large range of sample types – from extracellular vesicles4 that are barely above the limit of detection, to full Caenorhabditis elegans (C. elegans) on special large particle flow cytometers5 – and used in many different fields of research. As long as your particle of interest will pass through the fluidics system, it can be run on a flow cytometer. With the addition of fluorescence, through either specific dyes or fluorescence-conjugated antibodies, you can further interrogate the biology of your sample. Success in a flow cytometry experiment is generally an iterative process, with proficiency coming through experience. However, here are 10 tips that will help get you started on the right path. Stage 1: Planning 1. Know your biology The first key step in any flow cytometry experiment is to understand the cells you are working with. The source of the cells (tissue vs blood vs cell line, etc.) will dictate how your cells need to be processed for a flow cytometry experiment. In addition, you will need to know what targets you want to look at on your cells, the expected expression levels of the targets and where in the cell these targets are expressed. This knowledge will factor heavily in both your experimental and panel design. 2. Know your flow cytometer Understanding your flow cytometer is as important as knowing your biology, if not more so. Ultimately, they work hand-in-hand! Knowing the optical configuration (the lasers and filters available; standard vs spectral) will dictate which fluorochromes you can use on that platform and how many you can use. Some instruments may have the capacity for high throughput with automated tube or plate loaders. If your experiments will have large numbers of samples, this can be an important factor to consider as well. 3. Pyramid of panel design – it’s all about balance Panel design is the Sudoku of flow cytometry – you need to balance your target antigen expression/location with the available fluorochromes for that target with what your instrument is capable of, while simultaneously maintaining the highest sensitivity/resolution possible (Figure 1). Initial considerations in this puzzle are to optimize detection, and this is best achieved by placing the brightest fluorochromes on the targets that are most difficult to detect (be it because they are rare or have low expression levels). After this, it is important to consider spectral overlap of fluorochromes,6 spillover spreading error7 and which markers are mutually exclusive (markers that are expressed on different types of cells) – these will all impact your panel design. Panel design tools or expert advice can greatly simplify this process. The most important part of any panel design is to make sure that you rigorously test it before moving onto your experiments. Guide TEN TIPS TO GET YOU STARTED IN FLOW CYTOMETRY 2 Guide 4. Always include a viability dye Dead cells are death to a flow cytometry experiment (unless you are studying cell death). They generally have increased non-specific binding and increased autofluorescence. This can result in artifacts in your analysis and potentially decrease your resolution, depending on your fluorochromes and the autofluorescence signature. Viability dyes are now plentiful, many are fixable and they are available in a wide range of colors, so there is no reason to not include them in every experiment. Stage 2: Sample preparation 5. Titrate! Titrating your reagents is an important part of optimizing your detection. Titration is a simple process that requires limited reagents; the time it takes is worth the long-term savings you could make on reagents and the improvements in your experiment quality. To run a proper titration, serially dilute your reagents (generally 1:2), then use those dilutions to stain your cells. Don’t forget also to titrate your viability dye or other non-antibody reagents – titrations are not just for antibodies! Comparing the results obtained with differing concentration combinations will allow you to determine the ideal concentrations for your reagents with your cells on your flow cytometer.8 6. Single and ready to mingle (with your reagents) Having a single-cell suspension is the most critical part of your flow cytometry experiment.* While there are many steps that can be taken to attain single-cell suspensions, including filtering samples, oftentimes your buffers will be what make or break you. Understanding what buffer additives like ethylenediaminetetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or DNase can do will help you make decisions on how to formulate your staining buffers. Understanding the properties of your fixation reagents and how they work is both important to maintaining a single-cell suspension and in determining which fluorochromes you can use. Selection of a permeabilization buffer will depend on the location of your target antigen (i.e., cytoplasmic vs nuclear) and how many cells will be lost in the permeabilization process. Like everything else in flow cytometry, each of these should be tested in your system and titrated to determine the optimal conditions. Figure 1: Pyramid of panel design. Panel design in flow cytometry requires a fine balance to be struck between a number of important factors, including the target antigens, the fluorochromes available for those antigens and what your instrument is capable of. Credit: Aja Rieger. Brightness Options Instrument Capabilities Fluorochrome Choice Target Antigens Sensitivity TEN TIPS TO GET YOU STARTED IN FLOW CYTOMETRY 3 Guide *A special note on obtaining single cells: This is an art in itself and the exact method required to obtain them will depend on your sample source. If you are working with a tissue, you will need to determine your best method to get cells out of the tissue (mechanical, enzymatic, etc.) so that you can recover single cells without destroying too many.9 If you are working with an adherent cell line, you will need to consider the effects of cell detachment on your assay (e.g., is your marker of interest cleaved by trypsin?). Suspension cells (cell lines, blood, etc.) tend to be the easiest cells in this regard as they are generally already in a singlecell format. 7. Get control of your controls Controls are an integral part of any flow cytometry experiment. These include: • instrument controls (to ensure proper functioning of the cytometer) • single color controls (for compensation or spectral deconvolution) • fluorescence minus one (FMO) controls (gating controls) • isotype controls, positive controls, negative controls, unstimulated controls (protocol controls) • reference controls (to ensure consistency over time) Understanding when and how to apply these controls will aid in your downstream data analysis. Stage 3: Data collection and analysis 8. Getting your instrument set up and ready to go When starting your experiment, make sure your instrument is clean and that your sheath tank is full; steady pressure and quality fluidics are key to good data. When setting up your scatter and fluorescence detection, ensure at a minimum that your signal is on scale. If you wish to optimize your signal detection further, consider doing a voltage titration.10 9. Time to save some flow cytometry data! You’ve done the hard work to plan your experiment, optimize your protocol, controls and staining, and have your instrument ready to go. Now it’s time to acquire your data! As you get started collecting your cells, ensure you: • Collect a statistically relevant number of events. If you are looking for a cell that is one in a million, you’re never going to find it if you only collect 10,000 cells. This can be achieved by knowing your biology and setting a minimum collection number based on a specific gate, even if this is just your live cells gate. However, make sure you save all the events that go through the instrument. • Run on the slowest speed available on your flow cytometer to achieve maximum resolution. This will help to reduce the number of coincident events in your data file and will keep your cells centered in the stream. • Annotate your data. Include all the experiment information in the acquisition data files, including the marker name and sample information. Doing so will write the information into the FCS file* so it is available to anyone accessing your data (including you when, months from now, you may not remember exactly what was in each experiment). It will also make your data analysis much easier. *FCS = Flow cytometry standard.11 This is the standardized file format that flow cytometers generate. The FCS files hold all the relevant details about your acquisition, including the instrument the data was acquired on, the sample measured and the data obtained. Any details you add to your samples as they are acquired (e.g., markers and fluorochromes, tube name, treatment conditions) will be hard written into the FCS file, making them accessible to everyone. Ensure you are using the most up-to-date FCS version available. TEN TIPS TO GET YOU STARTED IN FLOW CYTOMETRY 4 10. The final stretch: completing the data analysis This is when you get to reap the benefits of all your hard work! Data analysis can seem overwhelming at times but following a standard workflow can help ease this.12 As you begin your data analysis journey, always start with a basic data cleanup. This includes removing spurious events with either a cleaning algorithm or by looking at fluorescence over time, removing debris, gating out doublets and putting your viability stain to good use. Your unstimulated, positive and negative controls will help to determine if your experiment “worked” (i.e., if the protocol allowed you to capture the information you were looking for). Your isotype controls will also be important at this point, allowing you to determine if your staining was “clean” (i.e., limited non-specific binding). Finally, you get to set your gates! Put your FMO controls to use to inform your gate placement. Bonus tip If you have access to a flow cytometry core facility, get to know the staff! They are your resident flow cytometry experts and can help to make your flow cytometry experiments as successful as possible. They will also have the best knowledge of the instruments you have available and will be helpful in planning your experiments. Sponsored by References 1. Wouters, M. What is flow cytometry? Technology Networks. Published December 15, 2020. Accessed January 10, 2024. https://www.technologynetworks.com/cell-science/articles/what-is-flow-cytometry-343977 2. McKinnon KM. Flow cytometry: An overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. doi:10.1002/cpim.40 3. Cossarizza A, Chang HD, Radbruch A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). Eur J Immunol. 2021;51(12):2708-3145. doi:10.1002/eji.202170126 4. Welsh JA, Arkesteijn GJA, Bremer M, et al. A compendium of single extracellular vesicle flow cytometry. J Extracell Vesicles. 2023;12(2):e12299. doi:10.1002/jev2.12299 5. Tjahjono E, Pei J, Revtovich AV, et al. Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases. Sci Rep. 2021;11:17733. doi:10.1038/s41598-021-97148-z 6. Roederer M. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry. 2001;45(3):194-205. doi:10.1002/1097-0320(20011101)45:3<194::AID-CYTO1163>3.0.CO;2-C 7. Nguyen R, Perfetto S, Mahnke YD, Chattopadhyay P, Roederer M. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry A. 2013;83(3):306-315. doi:10.1002/cyto.a.22251 8. Sheerar D. Titrating antibodies for flow cytometry. Published May 5, 2016. https://cancer.wisc.edu/research/wp-content/ uploads/2017/03/Flow_TechNotes_Antibody-Titrations_20170918.pdf 9. Reichard A, Asosingh K. Best practices for preparing a single cell suspension from solid tissues for flow cytometry. Cytometry A. 2019;95(2):219-226. doi:10.1002/cyto.a.23690 10. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 2006;69(9):1037-1042. doi:10.1002/cyto.a.20333 Guide TEN TIPS TO GET YOU STARTED IN FLOW CYTOMETRY 5 11. Spidlen J, Moore W, Parks D, et al. Data file standard for flow cytometry, version FCS 3.1. Cytometry A. 2010;77(1):97-100. doi:10.1002/cyto.a.20825 12. Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR. Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol. 2006;7(7):681-685. doi:10.1038/ni0706-681 About the author: Dr. Aja Rieger developed a love for flow cytometry during her undergraduate research days and never looked back. She received her PhD in comparative immunology at the University of Alberta, followed by a post-doctoral fellowship at the University of California- Berkeley, and has been the Flow Cytometry Core manager at the University of Alberta since 2015. She is highly involved in the field as the co-president of the Canadian Cytometry and Microscopy Association, as well as an instructor at the Annual Course in Flow Cytometry and Applied Cytometry Fundamentals of Flow. You can also find her educational content on YouTube (@ajarieger_flow). Guide