6 Tips For Applying The Right Statistical Test To Your Flow Cytometry Data
Flow cytometry data are numbers rich.
Data from experiments can be population measurements (percent of CD4+ cells, for example), or it can be expression level (median fluorescent expression of CD69 on activated T cells).
Many times, researchers are content to show histograms to illustrate their point after a flow experiment. This approach misses the opportunity to take that content rich data and extend the analysis into a statistical analysis.
To properly perform statistical analysis, the first step is to understand the hypothesis. The... Read More
7 Tips For Measuring And Reporting Apoptosis By Flow Cytometry
Cell death is a fact of biological life. How, when, where and most importantly, why cells die, can have huge biological consequences on the path an organism may take.
Apoptosis, or programed cell death, can result in a selective advantage for an organism. Fingers, for example, are the result of apoptosis of cells during development.
Next to immunophenotyping, measuring apoptosis using flow cytometry is one of the most common assays. It may be because of the many different... Read More
If You Don’t Know This About GFP, FITC, And PE, You Might Publish False Flow Cytometry Data
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 below illustrates the excitation and emission spectra of FITC under conditions of 488 nm excitation.
FITC’s emission maximum is... 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