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…
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…
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.…
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…
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…
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…
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…
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…
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…
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…
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.