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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…

What Is A Sample Injection Port

What Is A Sample Injection Port

By: Tim Bushnell, PhD

In flow cytometry, cells, in suspension are moved from the tube to the interrogation point and finally into the waste (or to be sorted, but that is a different story).  To do this, the fluidics components of the flow cytometry are required. The fluidics are comprised of a running (or Sheath) fluid, that runs through the system in laminar flow.  The movement of this sheath can be achieved by several mechanisms, the most common method using pressure provided by pumps. The second component of the fluidics is the sample injection port (SIP).  This is where the sample is pushed through…

What Is An Isotype Control

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

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…

Differential Pressure

Differential Pressure

By: Tim Bushnell, PhD

Differential pressure based flow cytometers currently dominate the market. These systems have two pressure regulators. The first is at a constant pressure that sets how fast the fluids runs at. The second is regulated by the investigator (like as shown on this LSR-II control panel). As the sample pressure goes from low, to medium, to high, the pressure on the sample increases. This results in the volume of the sample increasing (from ~15 ml/min to ~60 ml/min). The difference between the sample pressure and the sheath pressure is the differential pressure. This controls the width of the core stream and…

5 Keys To Writing A Shared Instrument Grant

5 Keys To Writing A Shared Instrument Grant

By: Tim Bushnell, PhD

I’m a scientist, not Perry Mason. I’m not a defense attorney or a detective. Why would I say that? Because well before March grant writers need to be a Perry Mason – make the case and defend it to have a successful Shared Instrument Grant (SIG). For those running core facilities (or Shared Resource Laboratories), then March is a critical time of the year, and we’re not talking basketball. A grant is a true story. This year’s deadline is March 21. It’s critically important in this tight funding climate to make sure nothing moves your grant from the “must-fund” to…

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Heather Brown-Harding, PhD

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Tim Bushnell, PhD

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Tim Bushnell, PhD

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