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FACSCalibur

FACSCalibur

By: Tim Bushnell, PhD

A type of flow cytometer manufactured and sold by BD Biosciences. This instrument was one of the first mass produced flow cytometers. The FACSCalibur is still prevalent in many labs around the world. While only a four color, six parameter analog system, this machine is stable and rarely requires service. It has gained a reputation as a “work horse” in core facilities. Recently, companies such as Cytek have begun refurbishing these analog instruments into digital models. This hybrid instrument produces a machine capable of acquiring data digitally, in FCS3.0 format, while still utilizing the reliable fluidics system that the Calibur…

Flow Cytometry Teaching

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

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

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/

Flow Cytometry Statistics

Flow Cytometry Statistics

By: Tim Bushnell, PhD

Understanding statistics and fow cytometry statistical analysis is critical to understanding flow cytometry data. One of the powers of flow cytometry is the fact that we generate large amounts of data that are amenable to statistical analysis of our populations of interest.  Using the standard set of statistical analysis tools allows for hypothesis testing and ultimately determining if there is statistical significance in the datasets. There are two basic classes of questions that are typically asked in flow cytometry.  The first class relate to changes in the number or percent of a specific population upon treatment or disease state.  A…

FACS Analysis

FACS Analysis

By: Tim Bushnell, PhD

Flow cytometry is the science of measure the physical and biochemical processes on cells and cell-like particles. This analysis is performed in an instrument called the flow cytometer.  FACS Analysis is the short-hand expression for this type of cell analysis The term FACS stands for Fluorescent Activated Cell Sorting, a term first coined by Len Herzenberg in the 1970’s, and later trademarked by Becton Dickinson. Since that time, FACS has come to be used as a generic term for all of flow cytometry, even though it is a specific trademarked term.

Spectral Profile And Spectral Viewer

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…

Phosphate Buffer

Phosphate Buffer

By: Tim Bushnell, PhD

PBS is the acronym for phosphate buffered saline. Phosphate buffer is one of the most common buffers used in biological research.  The phosphate serves as a buffer to keep the pH constant, while the saline is referencing the osmolarity.  Additional ions such as Ca2+ or Mg2+ , energy sources like glucose, or chelators such as EDTA can be added based on the specific needs of the experiment. There are many different formulations of PBS, based on the cell type and needs.  The most common, from Cold Spring Harbor is shown below, providing a pH of 7.4 with an osmolarity to normal…

What Is Autofluorescence

What Is Autofluorescence

By: Tim Bushnell, PhD

What is autofluorescence? Autofluorescence is the term given to describe the natural fluorescence that occurs in cells. The common compounds that give rise to this fluorescence signal include cyclic ring compounds like NAD(P)H, Collagen, and Riboflavin, as well as aromatic amino acids including tyrosine, tryptophan, phenylalanine. These compounds absorb in UV to Blue range (355-488 nm), and emit in the Blue to Green range (350-550 nm). The consequence of this autofluorescence is the loss of signal resolution in these light ranges and a decrease in signal sensitivity. Autofluorescence typically increases with cell size. Larger cells have more autofluorescence than small…

What Is Dynamic Range

What Is Dynamic Range

By: Tim Bushnell, PhD

Dynamic range is the total range of fluorescent values obtained from a particular flow cytometry assay. It is defined as the ratio of the largest possible fluorescent signal to the smallest possible fluorescent signal. The dynamic range can vary based on the application. For example, a cell cycle assay may have a dynamic range of only 1000 fluorescence units. Surface staining against CD3 may have a dynamic range of 10,000. There is some debate as to the largest dynamic range required for flow cytometry, with some estimates putting the largest required dynamic range at about 3.5 and others arguing for…

What Is A Fluorescence Minus One, or FMO Control

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 Sheath Fluid

What Is Sheath Fluid

By: Tim Bushnell, PhD

Sheath fluid is the solution that runs in a flow cytometer.  Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure.  The principles of hydrodynamic focusing cause the cells to align, single file in the direction of flow. Depending on experimental needs, different formulations of sheath fluid can be used. Many labs purchase pre-mixed phosphate-buffered saline from Leinco Technologies. Some researchers use Hepes-buffered saline.  This is particularly useful for high-pressure cell sorting as Hepes controls pH better at high pressure than phosphate buffers do. Finally, since…

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