Flow Cytometry

We like to orient everyone to the GCC facility protocols and procedures for the instrumentation so we like to meet all new users prior to them utilizing the facility on their own.

Any single cell or single particle suspension can be analyzed, including mammalian cells, bacteria, yeast, dissociated tissue and polystyrene beads. If your cells or particles are not in a single cell suspension, you cannot perform flow cytometry. Cells that are not from whole blood must be filtered. Persons working with adherent cells must make sure that their cells stay in a single cell suspension before and during the entire process. It is recommended to suspend cells in a product called Accutase, which can be purchased from its creator, Innovative Cell Technologies, or from many other sources, e.g., SigmaThermo Fisher, Millipore Sigma, BioLegend. Filtering alone is insufficient to prevent adherent cells from reforming clumps.

For analysis, samples should be brought in 12 x 75 mm round bottom style, polystyrene plastic Corning Falcon test tubes (catalog #352008). The same size tubes from other manufacturers may not necessarily fit the sample O-ring on the analyzer flow cytometers. For sorting, samples should be brought in 12 x 75 mm round bottom style, polyproylene or polystyrene plastic Corning Falcon test tubes (catalog #352063 and 352008, respectively) or in 15 ml conical tubes.

This Web site has a number of links to important and comprehensive protocols. Click on the Protocols & Useful Links section of the facility's Web site to access them.

For analysis, we recommend that you put 1 million cells in 500μl of FACS buffer into each test tube.

 There are different types of flow cytometry controls that are necessary every time an experiment is performed. Basic flow cytometry controls consist of negative and single color controls. Negative controls consist of cells alone or an isotype control. Single color controls are used for compensation of fluorochrome emission overlap. Single color controls must have sufficient signal to be able to adjust instrument settings (i.e. they MUST be visibly POSITIVE). Single color controls do not necessarily have to be the same antibody as the antibody of interest as it is the fluorophore that is being controlled for, not the antibody. If the conjugated antibody of interest is not brightly expressed, that antibody would not be an appropriate single color control. Some manufacturers sell beads that are designed to bind antibodies, creating the necessary single color controls that flow cytometry requires (Thermo Fisher OneComp eBeads, UltraComp eBeads and AbC Total Antibody Compensation Beads). Some persons may and should choose to prepare additional controls consisting of fluorescence-minus-one [FMO] controls. FMO controls determine how to analyze the sample, i.e., to assist with gate setting for positive and negative expression. They should be considered a must whenever accurate discrimination is essential or when antigen expression is relatively low. FlowJo's 'Daily Dongle' provides an explanation of FMO vs. Isotype Controls. FMO controls cannot be used to set compensation. Related to typical flow cytometry controls, positive controls are used to ascertain the activity of an antibody of interest and are required in certain situations. For example, you may want to prove that your cells do not express a particular antigen. In this situation, you should supply a different cell type that expresses the antigen to prove that the antibody is specific to the epitope of interest. Experiments run without all of the appropriate controls make the interpretation of the information difficult, and any resulting conclusions must be considered suspect.

It depends upon the dye's excitation and emission characteristics. The facility is equipped with a variety of lasers although it is not comprehensive. Look over the GCC flow cytometry equipment webpage for a summary table of the available lasers and details of fluorochrome detectors available on each instrument. In addition, check out the fluorescent spectrum viewers from BD Biosciences, Thermo Fisher, BioLegend and Enzo Life Sciences found in the Protocols & Useful Links.

It depends upon the dyes' emission characteristics and the instrument. Again, check out the fluorescent spectrum viewers from BD Biosciences, Thermo Fisher, BioLegend and Enzo Life Sciences found in the Protocols & Useful Links.

The primary shared resource laboratory room (CN4158C) does not currently accommodate any biohazardous specimens (BSL1 level). Specimens must be inactivated of all pathogens before they are brought into the facility. Commonly, 1-2% paraformaldehyde is used. The laboratory recommends liquid methanol-free EM grade 16% paraformaldehyde from Electron Microscopy Sciences. This can be diluted using PBS. (10 x 10ml glass ampules are $22.50). Users should be aware that Invitrogen has a variety of LIVE/DEAD Fixable Kits that use an amine reactive dye that allows users to fix cells and to subsequently discriminate live from dead cells in the original sample. Other than preserving cells, these kits have the added benefit in that they inactivate pathogens. The exception to the BSL1 rule is for the sorter flow cytometer, which is housed in a different area (CN4146C). That instrument and the procedures associated with its operation allow it to be capable of sorting/analyzing specimens at the BLS2+ level. Use of this instrument is currently provided as a service only. Please contact us for details.

Persons that have never sorted cells before must first contact facility personnel. They will ascertain the feasibility of your sort goal, provide you with guidelines associated with sorting cells and create an appointment for sorting with you.

 This section is taken pretty much intact from the FAQ's for Flow Cytometry Acquisition/Analysis on the University of Virgina's Flow Cytometry Facility's webpage formerly directed by Joanne Lannigan — Thanks, Joanne! Nonspecific binding can be due to several reasons: Too much antibody will increase the amount of nonspecific binding of your negative population, which will reduce the signal-to-noise ratio. You should perform titrations for every antibody you use to determine the optimum concentration. Nonspecific binding can also be due to Fc-receptor binding. Use IgG of the same species as your antibody of interest to block nonspecific binding. Incubate samples with a final concentration of 2 mg/ml of IgG for ~10 minutes at room temperature before adding antibodies. Using monoclonal antibodies specific for Fc receptors to block Fc-mediated binding can also help reduce background binding. The use of directly conjugated antibodies can also reduce the amount of non-specific binding. If your antibody is not commercially available as a directly conjugated antibody, there are a number of simple procedures with which you can easily conjugate your antibody:

1. Thermo Fisher offers conjugation kits for their Alexa dyes that are very simple to perform. In addition, they also offer Zenon labeling kits, which utilize fluorochrome conjugated Fab' antibodies directed against the specific isotype of your antibody. A simple 10-minute incubation is all that is required.
2. Other options include conjugation kits available from Prozyme, Inc. and the standard do-it-yourself protocols.
3. The use of a biotinylated antibody with a streptavidin fluorochrome conjugate can be a source of nonspecific binding in some cells. Biotin is a component of normal cellular metabolism, and as such, there will be truckloads of it within mammalian cells.  For a discussion of non-specific binding properties of streptavidin associated with its Arg–Tyr–Asp (RYD) tripeptide sequence which mimics the Arg–Gly–Asp (RGD) binding sequence of fibronectin. see Molecular Probes Handbook 7.6.

No data files are allowed to be stored on the computers in the lab. After running your samples you should immediately transfer your data files into BOX and subsequently transfer them to your own computer. Your data and your experiments are ultimately your own responsibility.

Scheduling for all equipment in the core facility is accomplished using iLab (Agilent CrossLab), a web-based electronic calendar.

A current schedule of the facility's fees can be viewed above. Users will be billed for the time that's actually used.