Flow Cytometry Laboratory Frequently Asked Questions

I am new to flow cytometry. Where should I start?: Contact the laboratory before purchasing reagents or preparing samples. We can help with instrument and reagent selection, experimental design, controls, and training. Please also review the facility policy. For some projects, researchers must add the facility location (250A Greene Hall) to their project’s Biological Use Authorization (BUA).
What is the difference between flow cytometry analysis and cell sorting?: Flow cytometry measures light-scatter and fluorescence properties of individual cells or particles as they pass through the instrument. Cell sorting adds the ability to physically collect selected populations for further experiments, such as cell culture or molecular analysis.
Which instrument should I use?: Instrument selection depends on your sample type, fluorescent labels, number of parameters, budget, and whether you need to collect cells after analysis. The NovoCyte Opteon provides spectral analysis, the CytoFLEX LX provides conventional analysis, and the CytoFLEX SRT provides cell sorting. The CytoFLEX LX currently has five operational lasers; its red laser is unavailable. We can help you select an appropriate instrument.
Can I operate an instrument independently?: Independent analysis is available after completion of instrument-specific training and authorization by the laboratory. Previous experience with another flow cytometer is helpful but does not replace training on our instruments. Please contact us to discuss training and access requirements.
Can the laboratory run my samples for me?: Assisted services are available. Contact the laboratory in advance to discuss your experiment, sample preparation, controls, scheduling, and applicable charges.
How do I schedule an experiment or request training?: Contact Rie Watanabe at rzw0041@auburn.edu. For a new project, include a brief description of your research goals, sample type, approximate number of samples, planned fluorescent labels, and preferred timeline. New cell sorting projects require consultation before scheduling.
How should I prepare my samples?: Preparation requirements depend on the sample and application. Samples should be compatible with the instrument’s fluidics and contain minimal clumps and debris. Confirm the appropriate buffer, sample concentration, volume, filtration, and storage conditions with the laboratory. Review the sample preparation guide before your appointment.
What controls should I bring?: Controls depend on the assay and may include unstained samples, single-stain controls for compensation or spectral unmixing, and appropriate biological or gating controls. Discuss your control plan with the laboratory before preparing samples.
Does the laboratory provide antibodies or compensation beads?: Researchers are generally responsible for purchasing the reagents and reference beads needed for their projects. Laboratory supplies are primarily used for demonstrations and user training and are not routinely provided for research experiments. We can advise on reagent and bead selection before you make a purchase.
Can I use an existing antibody panel on the Opteon?: An existing panel may be transferable, but it should be reviewed before use. Fluorochrome compatibility, reagent concentrations, sample autofluorescence, and reference controls can affect spectral analysis. Some panels will require adjustments or additional optimization.
What collection options are available for cell sorting?: The CytoFLEX SRT can simultaneously sort up to four populations into separate collection tubes. Alternatively, cells can be deposited onto glass slides or into individual wells of 96- or 384-well plates. Plate sorting options include single-cell deposition and index sorting. Discuss your collection format and downstream application with the laboratory in advance.
Can you sort human-derived, infectious, or genetically modified samples?: The laboratory operates under an institutional BSL-2 Biological Use Authorization. Sample acceptance depends on the material, associated hazards, proposed sorting procedures, and whether the work falls within the laboratory’s current authorization. Some samples may require enhanced containment or additional institutional review and approval before sorting can be scheduled. Please contact the laboratory early in your planning so that we can assess the requirements and determine whether the proposed work can be accommodated.
How much do services cost?: Please refer to the current service rate schedule. Charges depend on the instrument and level of assistance required. For projects involving substantial ongoing support or scientific collaboration, please contact the laboratory during grant planning to discuss budgeting and potential support arrangements.
Can you help with data analysis?: Consultation on gating, compensation, spectral unmixing, data interpretation, and presentation is available. Please discuss your analysis needs in advance.
How should I save and retrieve my data?: Follow the laboratory’s current data transfer and storage instructions. Researchers are responsible for retrieving their data and maintaining secure backups. Confirm the transfer method and file availability with the laboratory before completing your experiment.
When should laboratory personnel be included as coauthors?: Projects involving substantial contributions to experimental design, analysis, interpretation, or manuscript preparation may warrant co-authorship. Please discuss the anticipated contributions and publication expectations with the laboratory early in the collaboration. Authorship should reflect the contributions ultimately made and the applicable journal’s requirements.
How should I acknowledge the laboratory in a publication?: Acknowledge the Flow Cytometry and High-Speed Cell Sorting Shared Resource Laboratory, College of Veterinary Medicine, Auburn University, and include its Research Resource Identifier (RRID: SCR_025507). Please contact the laboratory if you need an instrument description or assistance documenting the methods used.
Does this laboratory provide diagnostic flow cytometry?: For diagnostic flow cytometry services, please contact the College of Veterinary Medicine’s Clinical Pathology Laboratory.