Frequently Asked Questions

What is Flow Cytometry?
Flow cytometry is a technology whereby a stream of cells is passed through a laser beam at high speed (thousands of cells/second). As each cell passes through the laser beam, light is scattered and detected by the flow cytometer. This allows counting of the cells in the sample. If the cell contains any molecules that fluoresce at the wavelength of the laser light, fluorescence is emitted; this allows counting of the cells with the property associated with fluorescence (for example, labeling with a fluorescent antibody). These data are used to count the types of cells in the specimen and to infer the characteristics of those cells.
How does Flow Cytometry work?
Light scattered in the forward direction is collected by the Forward Scatter channel (FSC) photodiode and provides information on the relative size of the particle (see diagram below). Light measured at a 90° angle to the laser excitation beam is collected by the Side Scatter channel (SSC) photomultiplier tube (PMT) and provides information on the relative granularity of the particle.
Separate fluorescence channels (FL-) detect any fluorescent light associated with the particle. The light is directed via a series of mirrors and filters to PMT´s. Filters determine the wavelength of light to be detected by the PMT, any unfiltered light is reflected by a dichoric filter and is channeled to the next fluorescent channel.
workflow for flow cytometry
Which machine should I use?
If your experiment requires 1-3 colors (3 different color antibodies) the FACScan should be sufficient. If you need to do a 4 color experiment or use APC then the FACSCalibur would be the instrument of choice. If you want to use more than 4 colors or sort cells the FACSAria would be the best choice.
What types of controls will I need for my experiment?
It depends on the type of experiment you are performing. For surface staining of cells, three controls are needed in an experiment. In each instance, the controls must undergo the same treatment (i.e., preparation) as all the tubes in an experiment. An Unstained Control is used to detect "auto-fluorescence" or background staining of the cells of interest. Auto-fluorescence can be a significant problem, particularly in systems that contain monocytes/macrophages, cultured cells, or activated cells. An Isotype Control (i.e., where an antibody is used that has the same immunoglobulin isotype as the test antibody, but a different specificity which is known to be irrelevant to the sample being analyzed) is needed to determine whether fluorescence that is observed is due to non-specific (e.g., due to Fc receptor on cells of interest) binding of the fluorescent antibody. Efforts should be made to use the same concentration of isotype antibody as the antibody of interest.
All events (cells) that display fluorescence above the threshold delineating the above two controls are considered positive for the marker of interest. Another type of control that can be used to determine positivity is termed fluorescence minus one or FMO. This control works like a isotype control without the actual antibody. FMO controls are set up by leaving out one of the antibodies in your staining panel. FMO controls are especially important when doing multiparametric flow cytometry (more than 4 colors). Isotype controls are important for experiments where "sticky" cells are stained (e.g. Monocytes, Dendritic cells, B cells etc.) whereas, FMO controls maybe best for "non-sticky" cells (e.g. T cells). The researcher needs to keep in mind that isotype control antibodies are not the exact same antibody and that they may have higher or lower background staining. Lastly, a Positive Control is highly desirable (if not always available) to prove that the test antibody is functional and to allow proper setting of the flow cytometer. The positive control should include cells known to be positive for the marker of interest. Another type of control is the Fluorescence Control, discussed in the next question.
What is Fluorescence Compensation?
Briefly, in any experimental system where two or more fluorochromes (multi-color analysis) are used to characterize the cells of interest, fluorescence compensation must be considered. Fluorescence compensation is the mathematical subtraction of the fluorescence due to one fluorochrome from the fluorescence due to another fluorochrome. This is necessary because emission spectra from two fluorochomes can overlap. For this reason, in a sample that contains (for example) both fluorescein and phycoerythrin (two of the most common fluorochromes), fluorescence due to FITC (for example) will be detected by the electronics that are set up to detect fluorescence due to phycoerythrin. If this were not subtracted from the phycoerythrin fluorescence, the latter would be spuriously high. To determine the correct amount of fluorescence compensation, cells stained with only one fluorochrome-conjugated antibody are analyzed individually. This must be done for each of the fluorochromes used in the experiment. On both the Facscan and FACSCalibur you will need to manually compensate you data. The FACSAria which is used for mulitparametric flow cytometry or cell sorting does have an automatic compensation feature.
Ask us about compensation beads when working on the FACSAria.
Is it important to have highly viable cells for Flow Cytometry?
Dead cells can non-specifically bind antibody and thus can appear to the flow cytometer as positive events. The flow cytometer can discriminate some dead cells based on their forward and side scatter characteristics, but cells that have recently died or are not healthy can lead to false positive results.
How can I discriminate dead cells from live cells?
Dead cells can be identified by the use of a vital dye. Also, cell viability can be measured through the use of dye exclusion methods, either prior to flow cytometric analysis by light microscopy such as trypan blue, or during flow cytometric analysis through the use of propidium iodide, 7-amino-actinomycin D (7-AAD), EMA, Hoechst or DAPI dyes. Please speak with the facility to determine which viability dyes will work best for your application.
I need help designing my experiment. Can the Core staff help me?
Yes. Please contact Brent Palmer Brent.Palmer@uchsc.edu for questions related to experimental design and technical aspects of cell sorting or analysis. Short question require no collaboration but we ask that collaborations be established for more in depth guidance.