A filter that allows light between a set wavelength to pass through and reflects light above and below the set wavelength. For example, a bandpass filter with a wavelength of 550/40nm would allow light between 530nm and 570nm to pass through, but reflect light below 530nm and above 570nm.
A filter that allows light over a set wavelength to pass through and reflects light above the set wavelength. For example, a shortpass filter with a wavelength of 450nm would allow light with a wavelength less than 450nm to pass through the filter, but reflect light higher than 450nm.
A filter that allows light over a set wavelength to pass through and reflects light below the set wavelength. For example, a longpass filter with a wavelength of 670nm would allow light with a wavelength greater than 670nm to pass through the filter, but reflect light lower than 670nm.
Counted by the Photomultiplier Tube (PMT) in the flow cytometer. Photons enter the PMT and the signal is amplified in the PMT when a photon strikes the anode and “knocks” of electrons. These electrons then hit a series of subsequent anodes, amplifying the total number of electrons of signal. The PMT then counts the total number of electrons and this is converted to the signal.
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 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/
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…
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.
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…
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…