Fluorescence Funny Business
When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb. What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the input light.
It is important to examine a fluorophore in terms of its excitation and emission spectra, which essentially indicate the probability that a molecule will emit a photon of a certain wavelength of light given an excitation photon of a given wavelength. Figure 1 (1) below illustrates the excitation and emission spectra of FITC under conditions of 488 nm excitation.
FITC’s emission... Read More
How To Do Phospho-Flow Cytometry
I often have researchers come into the core wanting to look at the activation and downstream signaling events that occur in different immune cells.
These events occur in response to signals such as cytokines, chemokines, various receptor ligands, and the engagement of the T cell or B cell receptors. The signaling events are also characterized by the initiation of several phosphorylation events.
Measuring Phosphorylation Events
When this is the case, I recommend that the researchers set up a phospho-specific flow cytometry, or phospho-flow, experiment. These types of experiments measure the phosphorylation state of intracellular proteins at the single cell level.
Phospho-flow allows for the analysis of many phosphorylation events, along with cell surface markers, simultaneously. These types of experiments enable the experimenter to resolve complex biochemical signaling networks in heterogeneous cell populations. Phospho-flow has been applied to numerous areas of biology, including antigenic stimulation and microbial challenge,... Read More
5 Mistakes Scientists Make When Doing Flow Cytometry Proliferation Experiments
Measuring cell proliferation can be done in a number of ways.
There is the below tried and true method of counting cells. This straightforward assay can help determine if the cells are proliferating and by comparing counts. Here, a researcher can determine that the experimental treatment is increasing cell growth.
A second method of measuring proliferation involves using a radioactive tracer like 3H-Thymidine. In this assay, the amount of the isotope taken up by the cells correlates to the amount of DNA synthesis, and therefore growth. Of course this requires using radioactivity and all that entails.
A third... Read More
How To Design Accurate & Effective Flow Antibody Panels (or, What’s An OMIP?)
I was at a meeting talking about the principles of panel design.
At the end of my talk, I had an investigator approach me and ask why he was not making progress on his 15-color panel that he started developing. So, I asked how long he’d been working on it.
That was his response. This might shock some of you but a month is not very long when it comes to designing an accurate and effective antibody panel for a flow cytometry experiment. Multicolor panel design requires a delicate balance of biology and physics. Understanding the biology of the system and the physics of flow cytometry are critical to success.
Antibody panel design (and flow cytometry experimental design in general) is a complicated process that can take a very long time. There are, however, some things you can do to simplify the process and shave weeks if not months off of your design time, including:
1. Knowing your biological question.
The driver in this whole process is knowing what the question is. This question will help determine... Read More
10 FlowJo Version X Hacks That Will Help You Publish Your Flow Cytometry Data
So you just got the most amazing results of your life and you can wait to show it off in lab meeting, or create the figure for a publication. Here are a couple of tips that will help you ensure that everyone else also sees how amazing your data is!
1. If you only have a few events, use the option to “show large dots”.
When you only have 4-5 events in a population, it can often be difficult to see. If you turn off the high resolution, you can see the data better.
Double click on a plot in the layout editor and under the specify tab, check the box for ‘Use Large Dots”
2. Make sure your axes are labeled properly and cleanly with something that makes sense.
Many cytomters have a default axis label like FL1, or 525/50, as two examples. Some scientists doing flow are savvy enough to use the $PnS keywords to enter in their own axis labels during acquisition.... Read More