Flow Cytometry (FCM) is a widely used technique for cell counting, fluorescence-activated cell sorting (FACS), cell cycle analysis, identifying cell populations in heterogeneous samples, analyzing cell characteristics and function, as well as measuring cell surface and intracellular molecules. Flow cytometry is an efficient high throughput technique, capable of recording multiple signals at once.
UV or Near UV Laser Line |
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Cat. No. | Product Name / Activity |
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5748 | DAPI |
Fluorescent DNA stain | |
5117 | Hoechst 33342 |
Fluorescent dye for staining DNA | |
Violet Laser Line |
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Cat. No. | Product Name / Activity |
6489 | Ocean Blue, SE |
Blue fluorescent dye; supplied as NHS ester | |
Blue Laser Line |
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Cat. No. | Product Name / Activity |
7121 | 7-Aminoactinomycin D |
Fluorescent DNA stain | |
5119 | Calcein AM |
Cell permeable compound; hydrolyzed to become fluorescent in living cells | |
5935 | H2DCFDA |
Fluorescent ROS indicator; cell permeable | |
6444 | MitoMark Green I |
Green fluorescent mitochondrial stain; cell permeable | |
5135 | Propidium iodide |
Red-fluorescent DNA stain; membrane impermeant to live cells | |
Green Laser Line |
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Cat. No. | Product Name / Activity |
5440 | FITC |
Green fluorescent dye | |
Yellow Laser Line |
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Cat. No. | Product Name / Activity |
6503 | Janelia Fluor® 549, free acid |
Yellow fluorescent dye; supplied as a free acid | |
6500 | Janelia Fluor® 549, Maleimide |
Cell permeable yellow fluorescent dye; supplied with a maleimide reactive group | |
6147 | Janelia Fluor® 549, SE |
Cell permeable yellow fluorescent dye; supplied as NHS ester | |
6502 | Janelia Fluor® 549, Tetrazine |
Cell permeable yellow fluorescent dye; supplied as a tetrazine for 'click' chemistry | |
7693 | MitoBrilliant™ Live 549 |
Yellow/orange fluorescent mitochondrial stain for live cells, Δψm dependent. | |
Red Laser Line |
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Cat. No. | Product Name / Activity |
5436 | Cyanine 5, SE |
Red fluorescent dye for the labeling of amines; supplied as NHS ester | |
7088 | Janelia Fluor® 646, Azide |
Cell permeable red fluorescent dye; supplied as an azide for 'click' chemistry | |
6993 | Janelia Fluor® 646, free acid |
Red fluorescent dye; supplied as a free acid | |
6590 | Janelia Fluor® 646, Maleimide |
Red fluorescent dye, supplied with a maleimide reactive group | |
6148 | Janelia Fluor® 646, SE |
Cell permeable red fluorescent dye; supplied as NHS ester | |
7700 | MitoBrilliant™ 646 |
Universal red fluorescent mitochondrial stain for both live and fixed cells. | |
7417 | MitoBrilliant™ Live 646 |
Red fluorescent mitochondrial stain for live cells, Δψm dependent. | |
Other |
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Cat. No. | Product Name / Activity |
7313 | Hoechst Janelia Fluor® 526 |
Fluorogenic, green-emitting DNA probe | |
6804 | Hoechst Janelia Fluor® 646 |
Fluorogenic, red-emitting DNA probe |
There are several different of types of flow cytometry including spectral flow cytometry, imaging flow cytometry, mass cytometry and cell sorting, but the principles remain the same. Pre-labeled cells are suspended in a buffer and are forced to flow one cell at a time through a flow cytometer instrument. A laser beam is directed at the single cell flow and a detector records the fluorescent and light scatter signals, which are then converted into a plot, allowing researchers to visualize their data.
Figure 1: Flow Cytometery Principles: There are three basic flow cytometer components: 1: a fluidics system that uses sheath fluid to force cells through a chamber one cell at a time; 2: an optics system, consisting of a laser that is focused at an interrogation point where the cells will be flowing one at a time. This laser excites the fluorophores conjugated to the labeled antibodies, with the resulting fluorescence emissions passing through a series of dichromic mirrors and optical filters. There are three common optical filters for recording the fluorescence emissions: longpass filters that allow wavelengths over 500 nm through; shortpass filters that allow wavelengths under 600 nm through; and bandpass filters, which allow a specific range of wavelengths through; 3: an electronics system, made up of photo multiplier tubes (PMTs) that capture the fluorescent emission (photons) signal and convert it into an electronic signal. In addition to fluorescence signals from fluorophore-labeled antibodies which identify the presence of a target, a flow cytometer detector also measures forward scatter (FSC) and side scatter (SSC) of the laser light signal. As cells pass in front of the laser, light is scattered, the pattern of this scatter is used to determine the size and nature of cells passing by. FSC is measured in the forward direction (the direction that the laser is traveling) and SSC is measured at a 90° angle. FSC and SSC readings are key controls to remove noise from debris in the cell solution. It is worth noting that the blue laser is usually used to measure light scatter.
There are several common laser lines used for flow cytometry, and these will govern which dyes are best to use for an experiment. Common laser lines are listed below.
General tips for selecting fluorophores:
Figure 2: Common Flow Cytometry Laser Lines.
Technological advances in terms of instrument capability and dye properties, have led to new types of flow cytometry applications including:
Use our spectra viewer to interactively plan your flow cytometry experiments. View the excitation and emission spectral data for all common flow dyes including Janelia Fluor ® dyes, TFAX (AF) dyes, Cyanine Dyes (Cy Dyes) and BDY (BODIPY®) dyes. Note: absorption spectra may be displayed instead of excitation spectra.
BODIPY® is a registered trademark of Molecular Probes.
Use our sister brands Novus' flow cytometry panel builder tool to design your experiment by enabling researchers to find validated antibodies that work with specific cytometers. Your panel can be exported and saved for reference. Advanced Features of this tool include a spectra viewer, spillover popups and antigen density selector.
Download the Bio-Techne Flow Cytometry eHandbook, full of practical advice, troubleshooting tips and conjugate options.
For a comprehensive list of flow cytometry workflow solutions, including flow cytometry-validated pre-conjugated antibodies, Cloudz™ cell selection kits, flow cytometry protocols and Milo™, a single-cell western blotting platform, which can analyze thousands of cells in a single run, visit our sister brand R&D Systems™.
Tocris offers the following scientific literature for Flow Cytometry to showcase our products. We invite you to request* your copy today!
*Please note that Tocris will only send literature to established scientific business / institute addresses.
This product guide provides a background to the use of Fluorescent Dyes and Probes, as well as a comprehensive list of our: