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Depending on the experimental design, many researchers will be doing complex assays that will require statistical analysis to determine if the hypothesis being tested is statistically significant or not. Unfortunately, many researchers go about this analysis the wrong way, resulting in spurious conclusions. The following points are guides to help think about the steps necessary in flow cytometry data analysis. 1. Before you start Define your hypothesis. This may sound simplistic, but understanding the purpose of the experiments is the first step in performing good statistical analysis. Stating the hypothesis will allow the researcher to choose the correct statistical test…

A laser type in a flow cytometer with a wavelength of about 560nm. The green and yellow laser are more effective at exciting PE and its tandems than the traditional blue laser. The yellow laser is also often used to excite the “fruit” dyes like mCherry. For more information, please review this journal article: Telford  W,  Murga  M,  Hawley  T, et.al. (2005). DPSS yellow-green 561-nm lasers for improved fluorochrome detection by flow cytometry. Cytometry. 68A: 36-44

After completing the perfect staining and cytometry run, the hard work begins – data analysis. To properly identify the cells of interest, it is critical to pull together knowledge of the biology with the controls run in the experiment to properly place the regions of interest that will be dictate the final results. Gating is an all-or-nothing data reduction process. Cells inside the gate move to the next checkpoint, while cells outside the gate – even by a pixel, are excluded. 1. Before beginning, know the populations of interest. While it may sound flip, knowing what cells are the target…

The laser type in flow cytometers with a wavelength of around 530nm. Standard “green” lasers are about 532nm, but vary between 530nm and 535nm usually. The green and yellow laser are more effective at exciting PE and its tandems than the traditional blue laser.

A laser with a wavelength in the UV range. Typically in flow cytometers, the UV laser has a wavelength of 350nm or 355nm. Some have a wavelength of 375nm.

Another very common laser after the “blue” and “red” laser in flow cytometers. A “violet” laser in flow cytometry typically is referred to as the 405 because most flow cytometers use a violet laser with a wavelength of 405nm. Pacific Blue and Pacific Orange are the most common fluorophores used with this laser, but Brilliant Violet fluors are gaining popularity.

The second most common laser in a flow cytometer after the “blue” laser. The “red” laser typically has a wavelength of 633nm, but new flow cytometers are starting to use a “red” laser with a wavelength of 640nm. The most common fluorophores excited and detected off this laser are APC, Alexa Fluor 660, Alexa Fluor 700, and APC-tandems.

The most common laser type in a flow cytometer. Typically, this laser has a wavelength of 488nm in flow cytometers.  In fact, the term “Blue” laser is often interchanged with “488” laser. Frequently used fluorophores excited and detected by this laser are FITC, Alexa Fluor 488, PE, PerCP, and their tandems.

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.