Flow cytometry is a technique used ubiquitously across most biology and biochemistry labs. The goal of flow cytometry is akin to that of a complex cell sorter. In most common uses for flow cytometry, fluorescence-activated cell sorting (FACS), cells are tagged with a fluorescent protein or dye and then sorted according to wavelength. In addition to emitted wavelengths, flow cytometers can also measure a variety of cellular characteristics, such as size and morphology. In doing so, flow cytometry can be used for a variety of applications, from measuring apoptosis and cell growth to oxidative stress, receptor expression patterns, and even DNA and RNA content.
In the latest episode of Lab Talks, we talked to two experts, Christiane Hassel, manager of the Flow Cytometry Core Facility at Indiana University, and Dr. Raif Yuecel, head of the Centre for Cytomics at the University of Exeter.
New technologies for flow
Lately, spectral flow cytometry appears to be all the rage in the field. In conventional flow cytometry, light is passed through dichroic mirrors and bandpass filters to select colors of light for detection using point detectors, like photomultiplier tubes. In spectral flow cytometry, gratings or prisms disperse light across a detector array, allowing the detection of a continuous, high-resolution optical spectrum.Dr. Yuecel noted that, over the last few years, “spectral flow cytometry has been making a big step forward from the hardware perspective” because it can measure the internal spectra signature of individual cell lines and allows for an increase in the number of dyes and panels.
Other tech advancements that the scientists thought were meaningful included spectral analyzers and spectral sorters that have increased their facilities’ capabilities, including expansion of panel design. Imaging sorters, like the ImageStream from BD, let you observe the sample, provide an image of your cells, and allow you to sort them. This adds a dimensional analysis to each sample by not only sorting based on scatter and fluorescence but also morphology as well as marker internalization and localization, among other things.
When Dr. Yuecel and Ms. Hassel were asked whether these technologies could one day replace microscopy entirely, both replied that the chances were very low; it’s nearly impossible to get the same resolution of a confocal microscope using flow cytometry. However, these new technologies will help flow cytometrists parse out cells and particles to sort, which will ultimately yield more quantitative data. In effect, it will always be more of a complement to microscopy. Dr. Yuecel finished by commenting on the potential for flow cytometry to provide such high resolution, it would make microscopy redundant: “Do we need that?”
New techniques and applications
Though not a new technology per se, omics platforms have been introduced into the flow cytometry workflow. Flow cytometry can now be combined with proteomics, transcriptomics, and genomics to provide an omics view at a single-cell level. With these new hybrid techniques, scientists have limitless possibilities when it comes to the types of samples facilities are now asked to run. Dr. Yeucel and Ms. Hassel have been asked to run nearly every biosafety level 2 specimen, from archaea, C. elegans (to sort neuronal cells), and microbes to arabidopsis plant nuclei, Drosophila cells (some of which have cell cycle properties similar to cancer cells), and sorting stem cells from snails found in New Zealand. They have also looked at endosomes, exosomes, and microparticles. As Dr. Hassel put it, “If it fits, it flows. It’s not just for immunology anymore!”
Flow cytometers have become somewhat portable, for example, they can be 00used on boats to measure changes in ocean microorganisms, many of which have naturally occurring fluorophores. These scientists are essentially tracking changes in the sea and oceans occurring due to climate change.
Optimizing the initial setup of a lab and maintaining it is key in providing a facility that’s able to produce high-quality research. To that end, both guests had a lot to say when it came to giving advice to new managers who are thinking of starting their own flow cytometry core facility. The main thing that stood out was how to handle space. Flow cytometers are relatively large machines; therefore, space is at a premium when initially conceiving a core facility. One must think of how many machines, what type of machines, and where to best place them to not only maximize the use of currently owned machines but for future growth as well. It’s essential that the instruments are catered to the kind of research that is being performed by scientists within the institution, and that air compressors and air quality be considered as well. Compressors can take up room and can be loud, so it’s better to have an internal pressure system, if possible. Humidifiers, dehumidifiers, and temperature control (with backup systems in case one fails) may be needed depending on the season. It’s also recommended that analyzers be divided from sorters to avoid possible contamination of the sorters. Height should also be considered to make sure there is enough room for biosafety cabinets.
Other considerations that the guests thought were important included having a dedicated booking system to make scheduling instrumentation use easy. Dr. Yuecel and Ms. Hassel noted that Clustermarket software was easy to set up and helped set their booking and invoicing. To that end, SOPs, protocols, and regulations (including those for booking and invoicing) were important to enforce from the start.
Relationships are also important in setting up and managing a lab. The guests advocated for becoming friends with everyone: PIs, grad students, managers of other core facilities, and even sales representatives for lab consumables. Ask for help when you need it and constantly ask questions. It also helps to have connections regarding financing capital for the facility. To that end, negotiating funding at the beginning is crucial; as Dr. Yuecel puts it, “Once it’s signed, you can’t go back.”
Local community involvement
At the end of the Lab Talk, Dr. Yuecel and Ms. Hassel encouraged all those using flow cytometry to become more involved in their local cytometry society and those around the world. In science, the best thing a researcher can do, no matter their position, is build their network and branch out for help and new ideas. It’s especially important when setting up a new facility, as getting the right people involved from the start will set you on the path to success and growth.
Dr. Yuecel and Ms. Hassel named an exhaustive list of communities, including:
- International Society for Advancement of Cytometry (ISAC): Home of CYTO University, which houses webinars, and holds an annual meeting. Its webpage can be used to find local associated societies and contact info as well.
- Great Lakes International Imaging and Flow Cytometry Association
- The Flow Cytometry Research Group of the Association of Biomolecular Resources Facilities (ABRF)
- European Society for Clinical Cell Analyses (ESCCA)
- Cytometry Society of Ireland
- Scottish Society of Cytomics
- The Annual Course in Flow Cytometry: A week-long course in the US.
- Expert Cytometry courses with Cheeky Scientist
- Royal Microscopical Society (RMS)