To help you get a basic understanding of flow cytometry, we offer some introductory explanations and provide you with a list of further resources.

Explaining Flow Cytometry

Below is an overview explaining the basics of flow cytometry.

What Is Flow Cytometry?

Flow cytometry uses fluorescent probes, usually labeled antibodies, to identify cells. Cells or particles tagged with fluorescent molecules enter the cytometer via a fluid stream. The molecules then pass by a laser, which emits a specific wavelength of light. The fluorescent signal is detected and amplified, then translated into an electronic signal, which is sent to the computer. Information about the relative size and granularity of a cell is recorded and the result is a visual presentation describing an individual or group of cellular events. If needed the cells or particles can be separated by sorting and further analyzed.

What is Cell Sorting?

Cell sorting differs from analysis since it involves the separation and isolation of various cell populations. Cells can be sorted using flow cytometry or magnetic labeling to differentiate and separate the cell populations. Cell sorting is similar to standard flow cytometry except that the stream is vibrated at a frequency that separates it into droplets. These droplets are given an electric charge according to their fluorescent profile and flow through an electric field that sends the charged drops of interest into a collection tube or plate while the unwanted cells are directed into a waste container.

How Flow Cytometers Work

This is a relatively complex question; since you can spend hours depending on how deep into the subject you want to go, we suggest some resources that will allow you to dive into the architecture of flow cytometry as far as you wish to go. 

Flow Cytometry Spectral Analysis Tools

One critical aspect of flow cytometry is for users to understand exactly what is being excited, the excitation spectra and associated emission spectra. This information is critical because the reagents used for fluorescent or confocal microscopy cannot always be used in flow cytometry. Fluorescent and confocal microscopes use mercury arc lamps that emit as much as seven lines of excitation; the sources used in flow cytometers are coherent single lines of excitation.

Within the last ten years, the number of available fluorochromes has dramatically increased. A fluorochrome (or fluorophore) is a fluorescent chemical compound that can re-emit light upon light excitation. If you are using a new fluorochrome that will be used in the core facility for the first time, you must provide the core director with the excitation and emission profiles so that we can determine the best excitation source and detection for the fluorochrome. Em-max and Ex-max (maximum emission and excitation, respectively) can be a starting point for making this decision, but having the excitation and emission spectra are preferred.

We request users wishing to use new fluorophores to double check these reagents with the Flow Core Director and staff; some reagents suitable for confocal or fluorescent microscopy may not be capable of being excited or detected by the cytometer.   Before investing time and money in your experimental design, please ensure that your reagents can be used with our flow cytometers. When in doubt, please consult the flow core director.

We offer two resources to help you gather the specifications of the fluorophore you wish to use.

Resources and Articles

We provide some further resources below to help you learn more about flow cytometry.

Academic, Research and Organization Links

Purdue University offers resources for everything related to flow cytometry, including an email archive for the flow cytometry listserv. The Scripps Research Institute  offers a similar library but on a smaller scale. The National Flow Cytometry Resource at Los Alamos National Laboratory  focuses on cutting-edge research into improving flow cytometry measurements. The International Society for the Advancement of Cytometry’s   (ISAC) mission is to facilitate the exchange of knowledge in the quantitative cell sciences.

For organizational resources, in addition to ISAC mentioned in the previous paragraph, there is the International Clinical Cytometry Society  (ISSC), an organization that proactively identifies regulatory, reimbursement and practice challenges affecting the cytometry community’s ability to deliver patient care in all geographies.

Vendor Links

A lot of important cytometry knowledge comes from the manufactures who create cytometry tools and devices:

Houston Methodist Links