The mission of the Georgia Cancer Center's Flow Cytometry shared resource is to provide access to state-of-the-art flow cytometers, support equipment, and associated software and services. Our instrumentation affords the ability to perform almost every published flow cytometry protocol.

There are several computer data analysis workstations available that facilitate the analysis, presentation and publication of flow cytometry data. There are 2 Mac stations and 4 PC stations, the  Mac's and the PCs are located in CN-4185C and CN-4158D.

Equipment Locations

  • Analyzers: CN-4158C (706-721-5468)
  • Cell Sorters: CN-4146A and CN-4146C (706-721-8473)

Equipment

Fluorophores

Useful Links

Contact Us

Flow Cytometry

Health Sciences Campus

Georgia Cancer Center - M. Bert Storey Research Building

Jeanene Pihkala, BS MT(ASCP)SH

CN-4158E

(706) 721-8880

jpihkala@augusta.edu

Service Rates

There is a minimum one hour charge for all assisted use which includes analyzers and sorters. All prices are listed per hour.

ANALYZER FLOW CYTOMETRY

Agilent Quanteon's, Penteon and Cytek Aurora

$45.00 unassisted/$75.00 assisted & training

ThermoFisher Attune NxT and BC CytoFLEX

$25.00 unassisted/$50.00 assisted & training

CELL SORTER FLOW CYTOMETRY

ThermoFisher Bigfoot

BD FACSAria II SORP

Mouse and Non-Infected - $75.00

Human and Infected - $85.00

Support Equipment

Computer analysis workstations

Facility Users - No Charge

Non-Facility Users - $10/hour

Consultation

No Charge

 

Publication Requirements

All users of the Georgia Cancer Center Flow Cytometry Shared Resource should acknowledge the facility in all publications resulting from work performed in the resource.

The types of acknowledgment are described below. Cases of disagreement or potential conflict arising from prior agreements (or more likely, lack of prior agreements) will be assessed and resolved by the administration overseeing the Shared Resource and/or office of Senior Vice President for Research.

ACKNOWLEDGMENT of Shared Resource

  • Independent use of the facility only (principal investigators, technicians, post docs, associates or graduate students who are trained operators, as defined in a previous section)

ACKNOWLEDGMENT Shared Resource AND SPECIFIC INDIVIDUAL(S)

  • Provision of routine procedures and methods by the staff

CO-AUTHORSHIP AND ACKNOWLEDGMENT of Shared Resource if all criteria are met as set forth in Augusta University's Policy on Authorship of Scholarly Activities

  • Significant involvement in the project, significant involvement in the interpretation of scientific data, involvement in the actual writing of a paper, use of original techniques, and/or novel experimental design by the facility stuff

SENIOR AUTHORSHIP, WITH PERMISSION GRANTED FROM THE INVESTIGATOR FOR USE OF MATERIALS, AND ACKNOWLEDGMENT of Shared Resource

  • Independent, original work by the facility staff

Frequently Asked Questions

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.

SamplesAny 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.
Dead cells are notorious for non-specifically binding antibodies. Inclusion of a viability dye such as DAPI, PI, 7-AAD, or TO-PRO-3 iodide, among others, in your assay will allow you to exclude the dead cell population from your analysis. You must make sure that your viability dye is compatible with the other fluorochromes in your sample. Also, cells cannot be fixed when using a viability dye as this will make them permeable to the dye and all the cells will appear dead. As mentioned previously, users should be aware that Thermo Fisher 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. Pathogens are inactivated in the process.

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 they reserve or the time that's actually used, whichever is greater.

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The Georgia Cancer Center's Flow Cytometry Shared Resource values your feedback. Please, take the time to complete this survey to let us know how your experience was and if there are any opportunities for improvement. Thank you for working with us and for sharing your insights.