SOEST FLOW CYTOMETRY
Example of a Flow Cytometry Data File
What is Flow Cytometry?
The Flow Cytometry Facility operated out of the School for Ocean, Earth Sciences and Technology at the University of Hawaii provides state of the art instrumentation for analyzing and sorting particles. Flow cytometers detect particles based on their size (scatter) and fluorescence (auto or imparted by a dye). These particles can be enumerated or simply characterized as to their scatter and fluorescence characteristics. Multiple populations can be separated visually and if desired, physically, based on these properties. Flow cytometers work ideally with cells in the 0.2 - 200 µm diameter size range and cell concentrations as low as 1000 cells/ml.
Flow Cytometer's Flow Cell
The SOEST Flow Cytometry Facility in the Pacific Ocean Sciences and Technology Building, Room 20 (POST 20) has two flow cytometers: one is a research-grade flow cytometer capable of sorting cells (Beckman-Coulter Altra) and the other is a bench top analyzer (Beckman-Coulter XL). These flow cytometers were purchased, in part, using funds from the National Science Foundation under MRI Grant No. 0215817. The facility also has an Olympus BX51 epifluorescence microscope with various filter cubes and a Beckman-Coulter Multisizer III particle counter/electronic sizing instrument.
Sorting Flow Cytometer (Beckman-Coulter Altra)
Benchtop (sea-going) Flow Cytometer (Beckman-Coulter XL)
The facility uses Beckman-Coulters Expo32 MultiComp software (PC compatible, up to Windows 2000, but NOT compatible with Windows XP), as well as Tree Stars FlowJo software (Mac compatible). Data files generated on the flow cytometers are archived on compact disc. For a fee, facility staff will analyze client data, reducing the listmode files to data in Excel or text-formatted spreadsheets.
picoplankton, including heterotrophic bacteria, analyses:
The Altra can be configured for quantitative analysis of marine
bacteria, including heterotrophic bacteria, Prochlorococcus and
Synechococcus. Picoeukaryotes can also be enumerated. Soon, we will
offer quantitation of marine heterotrophic eukaryotes.
Microplankton analyses: The XL can used to enumerate phytoplankton -- it is recommended for most samples. The Altra can also analyze and sort larger phytoplankton cells (< 20 um dinoflagellates, diatoms) using its large particle attachment (this service requires a minimum of 1 day's run time, as it requires significant modification to the optical compartment).
Cell Cycle analyses: Determination of the proportion of a cell population in G1, S, and G2, can be accomplished using either propidium iodide or Hoechst as the DNA stain.
Traditional Blood Cell Analyses:Antigen expression on blood cell subpopulations can also be assessed and subpopulations sorted, for non-pathogenic strains compatible with our laboratory's Biosafety Level 2 classification.
Other useful links for flow cytometry and fluorescence detection:
Purdue University Cytometry Laboratories(the premier site for information on flow cytometry and its applications)
Olympus Microscopy Resource Center(a great site to learn about many basic microscopy techniques)
Omega Optical (all you ever wanted to know about filters, as well as detailed spectral information for most fluorescent dyes)
Molecular Probes(their catalog is the best resource available to learn about the multitude of fluorescent dyes used in many applications)
Dr. Karen Selph
directs the facility. To schedule time on the instruments and for
consultation on sample preparation,
please call her at 808 956-7941 or email firstname.lastname@example.org.
Those who will need extensive use of the facility can be trained to operate the equipment.
The facility is operated on a re-charge basis. It is available to faculty, staff and students at the University of Hawaii, as well as to non-UH users. Should scheduling conflicts arise, priority will be given to UH users. The charges are as follows:
Actual sample run-time with technician assistance
$75/hour Actual sample run-time with no technician assistance*
$50/hour Off-line sample analysis by a technician
The minimum time charged is 15 min (1/4 hour).
*In order to be qualified for running samples with no technical assistance on the Altra flow cytometer, the user must have spent a minimum of 2 hours technician-assisted run time for training and be deemed by the facility director to be qualified to use the instrument un-assisted. The XL flow cytometer can be used with more minimal training.