5 – Flow Cytometry

I’ve been in the world of flow cytometry and cell sorting for a very long time. Now, don’t worry, this isn’t going to be some lament about the “good old days.” Well, maybe just a little. But there will be helpful takeaways, I promise. I was trained by the incredible staff in the core facility at the University of Arizona. When I moved to UC Davis and proceeded to start up a core facility, there were very few resources to guide me. No Google communities and LinkedIn groups. Email was new, there was the Purdue Cytometry List, but that was…

As with all complex technology there are many different levels of education that users should avail themselves of. Basic instrument operation This level of education is akin to learning how to drive a car.  At this level, the focus will be on how to put the sample on the instrument, how to adjust voltage and collect data.  Specific policies of the facility should also be covered. Advanced instrument operation At this level of education, the focus should be on advanced techniques on the instrument.  This would cover basic troubleshooting – identifying problems and how to solve them.  Advanced training should…

Flow cytometry can be an intimidating tool for the new cytometrist.  There are many sources that one can turn to for training and education. Local Shared Resource Lab Manager If you institute has a shared resource lab (a ‘core facility’), look in what training they provide.  This is your first line of training experience.  Here you can learn about the policies of the facility, how to operate the machine and how to design or analyze your experiment.  These SRL managers and staff have extensive experience with flow cytometry and can help guide you through the steps to get good, high…

An implementation of biexponential scaling published by the Herzenberg lab at Stanford. The biexonential scale is a combination of linear and log scaling on a single axis using an arcsine function as its backbone. The “logicle” implementation of biexponential was implemented in many popular software packages like FACSDiva and FlowJo. Other types of biexponential scaling exist, including Hyperlog. Biexponential scales are more generally referred to as hybrid scales and include other variations like lin/log or log with negative. More information on logicle sclaing can be found here: Parks DR, Roederer M, Moore WA. (2006). A new “Logicle” display method avoids…

Flow cytometry is a complex technology that requires understanding of sample processing, data acquisition and data analysis.  An individual experiment can take a dozen hours to prepare, hours to collect and days to analysis.  This is why flow cytometry training is critical in understanding and optimizing the use of this technology in the research or clinical setting. This technology has evolved rapidly over the last 20 years, with changes at every level.  Practices that worked for 2-4 color flow cytometry need to be reevaluated when moving to 6-10 color flow cytometry.  Also, with the development of new fluorescent dyes, new…

If you’d like a job in a flow cytometry core or lab, ExCyte can give you all of the training you need. Combined, our instructors have over 100 years of flow cytometry experience. If you are interested in researching available flow cytometry jobs, there are number of online resources that post open positions including: http://www.cyto.purdue.edu/flowcyt/jobs.htm  http://www.chromocyte.com/educate/Positions-Available

Individuals can now be certified in flow cytometry by taking the International Cytometry Certification Exam, which is jointly managed by the International Society for the Advancement of Cytometry (ISAC) and the International Clinical Cytometry Society (ICCS).  This exam covers the general principles of cytometry in a multiple-choice format.  Individuals who pass this test are allowed to use the termed ‘Certified Cytometrists’ as part of their credentials More information can be found here: http://cytometrycertification.org/

Every fluorophore has a unique excitation and emission profile which is usually displayed on a spectral viewer, or spectral graph. The combination of the excitation and emission profiles is the fluorophore’s spectral profile. Every fluorophore has a peak excitation wavelength (the wavelength at optimal excitation) and a peak emission wavelength (the wavelength of optimal detection). Each fluorophore will also have a much larger range of excitation and emission wavelengths at reduced optimization. This “curve” is what is displayed on a spectral viewer. The spectral profile of a fluorophore is used to determine the excitation and detection efficiency at any given…

The Fluorescence Minus One Control, or FMO control is a type of control used to properly interpret flow cytometry data.  It is used to identify and gate cells in the context of data spread due to the multiple fluorochromes in a given panel. An FMO control contains all the flurochromes in a panel, except for the one that is being measured.  For example, in the four color panel, there would be four separate FMO controls, as shown in the table below. The FMO control ensures that the any spread of the fluorochromes into the channel of interest is properly identified.…

Do you know what an isotype control is? Isotype refers to the genetic variation in the heavy and light chains that make up the whole antibody moiety. In mammals, there are 9 possible heavy chain isotypes and two light chain isotypes. Every antibody will have a specific isotype, and this is available on the technical information spec sheet. For example, you might have an antibody with an isotype of IgG1, kappa. This indicates the heavy chain is of the IgG1 isotype. Where things get interesting is that these isotypes can have different non-specific binding affinity to cells, which has lead…

Titration is the process of identifying the best concentration to use an antibody for a given assay. While the vendor will provide a specific concentration to use, this may not be appropriate for your assay. Performing titration is a simple process: fix the cell concentration, the time of incubation, the volume of reaction and temperature. The below data will help you understand what is titration. The graph displays an antibody from Leinco Technologies () that was used to stain 1×106 cells for 20 minutes on ice. To identify the best concentration to use, the modified Staining Index was calculated (see…

ExCyte chooses to train people in flow cytometry because we know what it’s like to feel the pain of ruined experiments. No one enjoys wasting thousands of dollars on reagents and priceless amounts of instrument and personnel time. This is especially true when grant funding comes into play. No one wants to miss out on a grant simply because the technology is too complex to master alone in a vacuum. How much time, money and resources are wasted by half-trained individuals trying to operate complex cytometers sold as pushbutton washing machines? Every vendor sells the dream that their instrument can…