The field of flow cytometry is seeing various trends in instrumentation–on one end are smaller, more compact and more powerful instruments for the high end needs of researchers, and on the other end are easier to use instruments that make flow cytometry more accessible to researchers.
Leading the way for 2015 would have to be the Beckton-Dickenson Fortessa X50. This instrument, as reported at Cyto2014, was able to do over 20 fluorescent parameters. This offers the potential for high-parameter (Mass Cytometry level) data using fluorescent antigens. If this technology is expanded to cell sorting, it will allow better isolation of even more complex subpopulations.
The Attune NxT from Life Technologies is a system using acoustical focusing. Coupling the power to run at very high flow rates with lower spread of the CV’s compared to traditional flow, it uses up to fourteen fluorescent parameters. Exploring the power of this kind of system will be novel and it has a very small footprint as well.
Following the trend to use volumetric systems for fluidics, which helps drive the size and complexity of the instruments down comes the NovoCyte from Acea Biosciences, with three lasers and 13 fluorescent parameters, this is another instrument to consider for the lab tight on space.
Originally built by a Chinese company Xitogen, Beckman-Coulter acquired them and rebranded the instrument as the CytoFLEX. This small system was recently reviewed by Ryan Duggan. The system collects a second side scatter parameter off the 405 nm laser, which is potentially useful for those interested in micro particles. Also, as described in Ryan’s blog, this system uses APD rather than traditional PMTs.
Cell sorting has traditionally been the domain of large, complex machines. However, with the release of the Propel Avalon (now Bio-Rad S-3 cell sorter), the door has been open for cell sorters that are small and easier to use. Leading this trend is the Viva by Cytonome. This is a a true one-color bench top sorter at under $100,000 that will be a boon to those research groups using GFP as a marker in cell transfection.
The Tyto from Miltenyi-Biotech is an intriguing new cell sorter based on microfluidics replaceable cartridge. It has a small footprint, making it ideal for labs tight on space. Second, it does not use electrostatic deflection for sorting–thus reducing the biosafety concerns of aerosolized cells. While it can only sort one population on a time, since the system is low-pressure, sequential sorting is possible. Of the newer generation of smaller sorters, this has the most fluorescent parameters, which allows for isolation of more complex populations.
Fluorescent nanocrystals have been both a boon and a bane for flow cytometrists. On the manufacturing size, they have difficulties in consistency and development of the best chemistry to link these organic crystals to biological tags. However, they expand the spectrum of what can be measured. For years these have been available from only a few vendors. Trilite nano crystals by QuantaCyte are an interesting new entry into the nanocrystal market. You can be sure we’ll be looking at how these perform compared to the traditional QDotos and eFluor Nanocrystals.
The Brilliant Violet dyes, developed by Sirigen and marketed by Biolegend and Beckton-Dickenson, remain the most exciting addition to the flow cytometrists toolbox. Brighter, with fewer issues compared to nanocrystals, they are rapidly replacing the nanocrystal to expand fluorescent. With the expansion of these fluorochromes into the ‘Brilliant Ultraviolet’ arena, cytometrists now have a reason to need a UV laser. True 18 fluorescent parameter analysis is becoming a reality with this expansion.
Another interesting area of development to watch will be the ability to measure RNA levels by flow cytometry. There are several companies marketing products in this space–all of which will allow the ability to measure mRNA levels in a phenotypic manner. eBioscience’s PrimeFlow RNA detection system and Advanced Cell Diagnostics RNAScope are two systems we’ll be watching closely.
One of the most tedious tasks in flow cytometry is panel design–searching databases and determining which antigen/fluorochrome combinations are available and will work on your instrument of choice. The impending update and release of Fluorish 2.0 will be a great tool for designing antibody panels and keeping a virtual inventory of your antibodies. As is the newcomer in this space–Fluorofinder. Both of these programs are essential for complex panel design and should be in every cytometrist’s toolkit.
Updates and improvements to data analysis software are always of interest. Some, like the release ofDIVA 8 (Beckton-Dickenson), were driven because of the changes in PC operating systems. Others, like FlowJo Enterprise, are designed to help with the complex analysis of large datasets.
Recent releases to Cytobank, including the integration of viSNE and channel-by-channel coloring of dot plots, are some intriguing new analysis tools that have been added to this freemium software package. For those performing CyTOF analysis, Cytobank and the SPADE platform remain one of the main tools for analysis of mass cytometry data.
Not to rest on their laurels, DeNovo’s team is hard at work on FCS Express 5. I am hearing some very good things about this package from the beta testers, including integrated statistical analysis platform. This would be a great boon for researchers and their workflow–performing the flow analysis and move the data seamlessly into a statistical analysis package. Finally, Flow Logic by Inivai remains a strong software analysis package. We’ve found it to be similar to FlowJo in data analysis logic with a more integrated GUI.
Competition leads to innovation–and that is good for the end user. Faster, cheaper, and easier instruments and software continue to be developed in the field, which can only serve to improve the researchers experience and ultimately data.