What is flow cytometry?
Flow cytometry is a method for the measurement of relative size, granularity, and fluorescence of single cells in a population of cells suspended in a liquid medium. Samples are stained with specific fluorescently-labeled reagents (e.g., antibodies). The labeled cells are illuminated by a 488 or 635 nm laser, and the fluorochrome emission (fluorescent intensity) and light scatter (cell size and complexity) are recorded.
The flow cytometer
The UIC has a Becton-Dickinson FACSCalibur Flow Cytometer. This flow cytometer (June, 2000 mfr. date) is a four-color, dual-laser, bench-top system capable of cell analysis using forward scatter, side scatter, and detection of fluorescence in four distinct color regions: > 670 nm (deep red), 653-669 nm (red), 564-606 nm (orange), and 515-545 nm (green). The instrument has a Mac G4 host computer, and most instrument functions are computer controlled. The instrument also has two lasers for exciting fluorochromes: an argon laser, which emits sapphire-colored light at 488 nm, and a red diode laser emitting light at 635 nm.
What fluorochromes are commonly used with this instrument?
Fluorochromes commonly used with these lasers include fluorescein, thiazole orange, acridine orange, phycoerythrin, propidium iodide, allophycocyanin, pharmingen/BD's phycoerythrin-Cy5 (Cy-Chrome) and peridinin chlorophyll protein (PerCP). Please note that UV and green excited dyes such as DAPI and rhodamine and its derivatives cannot be used with this cytometer.
Whom do I talk to at the UIC about flow cytometry?
Call Mark Townley at 603-862-0632 or email mtownley@cisunix.unh.edu for more information.
How do I submit samples for analysis?
You need to make an appointment with Mark Townley. Please make an appointment at least 24 hours BEFORE you'd like your samples run. If you are late or miss an appointment, you will still be billed for the entire reserved time, unless you cancel at least 24 hours in advance.
We currently do not train anyone outside of the UIC on the instrument, so all samples are run by a UIC technician. All samples to be run should be non-toxic, non-infectious, non-pathogenic, and fixed. The UIC reserves the right to refuse to run a sample if there is a concern about the toxicity and/or safety of the sample. For more on the University of New Hampshire's flow cytometry policy, please refer to the University's Biological and Chemical Safety Plan.
A sample submission form should be filled out for all samples prior to submission.
What size cells can be analyzed? In what concentration?
The FACSCalibur Flow Cytometer is best suited for the analysis of aqueous suspensions of cells or particles with diameters between 1 and 50 um (microns). It is important that you filter aggregated samples with 70 um nylon cell strainer (made by Falcon, part number 352350) before running them on the instrument. If cells have aggregated to the point that you can see them in the sample tube (and a vortex mixer does not break up the clumps), the sample will plug the flow cell on the instrument.
Samples should ideally contain 1 x 106 cells or particles per mL.
Samples should be submitted in capped 12 x 75 mm Falcon brand 5 mL polystyrene round bottom tubes (part number 352003).
How much sample do I need?
Even though sample consumption can be varied between 12 uL/min and 60 uL/min (allowing analysis of relatively small samples), please submit a volume of at least 1mL.
Anything else I should consider?
To make conclusions about your experiment, the proper controls are critical. Make sure you have positive and negative controls. You will always end up needing one more control than you have; so when in doubt, prepare more controls then you think you'll ever need. A negative control is an unstained sample (autofluorescence!!) or a sample stained only with the secondary, if this is the system you are using. If you have samples stained with more than one fluorochrome per test tube, bring in plenty of sample stained with each fluorochrome individually. This is for compensation purposes. For an explanation of this procedure, see Mario Roederer's website on compensation.
The UIC staff will provide as much technical support, advice, and direction as possible. However, the UIC is not responsible for problems caused by poor sample preparation or flawed experimental design.
Helpful sites and links
- Flow Cytometer Facility at UCONN
- Purdue University Cytometry Labs
- International Society for Analytical Cytology (ISAC)
- Flow Cytometry on the web
Good Books on Flow Cytometry
- Flow Cytometry: First Principles by Alice Givan (really good introduction)
- Practical Flow Cytometry by Howard Shapiro
- Flow Cytometry: A Practical Approach by Michael Ormerod (good information on DNA)
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