5 – Flow Cytometry

Logicle Scaling

By: Tim Bushnell, PhD

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 Teaching

By: Tim Bushnell, PhD

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…

Flow Cytometry Jobs

By: Tim Bushnell, PhD

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

Flow Cytometry Certification

By: Tim Bushnell, PhD

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/

Spectral Profile And Spectral Viewer

By: Tim Bushnell, PhD

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…

What Is A Fluorescence Minus One, or FMO Control

By: Tim Bushnell, PhD

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.…

What Is An Isotype Control

By: Tim Bushnell, PhD

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…

What Is Titration

By: Tim Bushnell, PhD

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…

How To Train Your Personnel

By: Tim Bushnell, PhD

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…

The Importance Of Teaching With Integrity And Passion

By: Tim Bushnell, PhD

Flow cytometry education has grown phenomenally in response to more sophisticated instrumentation, growing demands for more sensitive, high-speed and multi-parameter flow. Specialized training is critical to any flow lab competing in today’s global marketplace. The key is to find the right trainer. As a core director or lab manager, how can you tell if the training course you’re interested in is both credible and relevant in today’s fast paced and constantly evolving market? Some tips and tricks: 1. Ask for references. Ask for names of people who teach the course. Are they in a modern and competitive lab or business? Are…

The Jablonski Diagram

By: Tim Bushnell, PhD

A Jablonski diagram illustrates the electronic states of a molecule as well as the transitions between them. These states are arranged vertically by energy and grouped horizontally by spin multiplicity. Nonradiative transitions are indicated by straight arrows and radiative transitions by squiggly arrows. The vibrational states of each electronic state are indicated with parallel horizontal lines. For flow cytometry, its important to note that the energy of the emission is usually less than that of the absorption. As such, fluorescence normally occurs at lower energies or longer wavelengths.