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Flow Cytometry Resources & FAQ
Jump to: Links of InterestFAQsFlourophoresSOPs
Key Resources
Current Protocols in Cytometry and Current Protocols in Immunology are "best-practices" collections that distill and organize the techniques of peer-reviewed protocols for flow and image cytometry and immunological methods.
Purdue Cytometry ListServ
A valuable forum for the discussion of many aspects of flow cytometry is the Purdue
Cytometry Discussion List, which has an international following.
Persons may search the discussion archive. To subscribe to the discussion, visit this website. To post an email to the group, send an email to cytometry@lists.purdue.edu.
Links of Interest
Fluorescence Spectrum Viewers
Antibody Cross-Reactivity Resources
Vendor Links
- Cytek Biosciences (flow cytometers and antibodies)
- Agilent (flow cytometers)
- Thermo Fisher (Attune flow cytometers, Bigfoot sorter, antibodies, etc. - Invitrogen/Molecular Probes/eBioscience/Qdots)
- BD Biosciences (flow cytometry reagents)
- BioLegend (antibodies, reagents, etc.)
- Jackson ImmunoResearch Laboratories (quality secondary antibodies)
Paid Software Links
Free Software Links
- MFI Flow Cytometry Software - Eric Martz
- Bioconductor - an open source software (not specifically for flow cytometry) that uses the R statistical programming language and has some flow cytometry modules
Frequently Asked Questions
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We like to orient everyone to the GCC facility protocols and procedures for the instrumentation prior to them utilizing the facility on their own.
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Scheduling for all analyzers and the Miltenyi Tyto is accomplished using iLab (Agilent CrossLab), a web-based electronic calendar.
For sorting, new users must first contact facility personnel (flow@augusta.edu). They will ascertain the feasibility of your sort goal, provide guidelines associated with sorting cells and create an appointment for sorting with you.
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To check compatibility of your dye please visit the Flow Cytometry page for the instrument configuration. For panel planning assistance please see the Links Of Interest section above or email flow@augusta.edu for assistance. -
No data files are allowed to be stored on the computers in the lab. After running your samples, the data files should be immediately be transferred to BOX. Your data and your experiments are ultimately your own responsibility. -
Any single cell or single particle suspension can be analyzed. All samples mst be filtered before running on the machine. If your samples are prone to clumping (adherent cells, tumors...) filtering may not be enough and you will need to include extra additives (DNAse, EDTA, Accutase...etc) to prohibit clumping. -
For analysis, samples can be brought in 12 x 75 mm round bottom polypropylene tubes, eppendorf tubes or standard sized culture plates. For sorting, samples should be brought in eppendorf tubes, 12 x 75 round bottom polypropylene tubes or 15mL conical tubes. -
The primary shared resource laboratory room (CN 4158C) can currently accommodate biohazardous specimens up to BSL1. Please discuss with core staff the potential for unfixed samples. Due to the biosafety level all users MUST wear the appropriate PPE (lab coat, gloves and safety goggles). There are extra lab coats outside the door for urgent use. Specimens should be inactivated of all pathogens before they are brought into the facility. Please see us for recommended fixation techniques. The sorter (ThermoFisher Bigfoot), which is housed in a different area (CN 4146C) can handle up to BSL2+. Use of this instrument is currently provided as a service only. Please contact us (flow@augusta.edu) for details.
Fluorophores
When performing multicolor flow cytometric analysis, a major factor in the success of the analysis is the choice of which antibody to use with which fluorochrome. There are often many correct combinations possible. A number of factors need to be considered in making choices.
The chart below shows the staining pattern of the same monoclonal antibody conjugated to 12 commonly used fluorochromes. This chart details the tremendous differences observed among different fluorophores; and it should be used as a guideline for the relative intensities of various fluorophores run on the core's cytometers.
Another great fluorochrome brightness chart and overall multicolor experiment setup reference can be found in this BD Biosciences Application Note.
This document provides more details about commonly used fluorophores.
Autofluorescence is a major concern in basic research flow cytometry environments, and no section on fluorophores can be considered complete without recognizing the role that cellular autofluorescence plays.
Individual cell populations have characteristic levels of autofluorescence (fluorescent signals generated by the cells themselves). Mammalian cellular autofluorescence, at least in lymphocytes, comes predominantly from pyridine and flavin nucleotides. Pyridine nucleotides are excited most efficiently by UV light (~340 nm) and have an emission maximum in the blue range at 450-470 nm. Flavin nucleotides are the primary issue in flow cytometry laboratories because those molecules are excited in the cyan-blue range (430-500 nm) of the color spectrum, which is where the flow cytometer's primary lasers emit light (488 nm). When excited, flavin nucleotide's emission (530-550 nm) is the same emission range as FITC/eGFP (green), PE (orange) and, to a lesser extent, PE-Cy5/PerCP (red emission) and PE-Cy7 (far red emission). The take-home message is that while autofluorescence is observed in all fluorescence channels, it decreases dramatically at longer wavelengths (>600 nanometers).
- For cell types that exhibit high levels of autofluorescence, using a dye with a longer emission wavelength (e.g., APC, APC-Cy7) often provides an excellent signal-to-noise ratio.
- For cell types that are not very autofluorescent, such as fresh cells, the improved separation seen with long-wavelength excitation is less apparent. In those cases, conjugates such as FITC and PE can be used.
Please see this reference for additional information on autofluorescence
SOPs
- Quanteon and Penteon SOP
- Troubleshooting
- Aurora SOP
- Data Management Procedure
- Troubleshooting
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