Flow cytometry, typically using fluorescent probes which
bind to specific cell associated molecules, allows measurements of
various phenotypic, biochemical and molecular characteristics of individual
cells (or particles) suspended in a fluid stream. As the cells flow
past a focused laser beam of appropriate wavelength, the probes fluoresce
and the emitted light is collected and directed to appropriate detectors.
These detectors, in turn, translate these light signals into electronic
signals proportional to the amount of light collected. Information
regarding the relative size and granularity of a cell, for example,
is also obtained as these characteristics influence the way in which light
is scattered as the cell passes through the laser beam.
The use of flow cytometry at TSRI can be divided into two broad
categories, analysis and cell sorting.
- The ability of flow cytometers to evaluate cells at an extremely
rapid rate (e.g. up to 100,000 events per second) makes this technology
ideally suited for the reliable and accurate quantitative analysis
of selected physical properties of cells of interest. The sensitivity
of these instruments for detecting the presence of molecules expressed at
low levels is impressive; given high quality cell preparations
and reagents, as few as 100 molecules per cell may be detected.
- One of the properties of the larger flow cytometers is the
ability to electronically deflect cells with preset, defined properties
into a separate collection tube. For cell purification, flow cytometry
is especially well suited for applications requiring high purity.
Because multiple fluorochromes (e.g. up to ten distinct fluorescent probes
reacting with different cell associated molecules) can be assessed
simultaneously, cell sorting by flow cytometry can separate complex
mixtures of cells on the basis of multiple marker expression.
The Flow Cytometry Core Facility is located on the first floor of the Immunology building and encompasses approximately 900 square feet of space. Additionally, we have two satellite labs. One is located at Science Park 3030
room 2030 and the second is in the MEM building in room 230. The facility is extremely well equipped for both analysis and cell sorting. The facility is available for use by outside groups on a special basis (please contact the
Flow Staff for information as the rates will differ from those posted on the website). A brief description of the cytometers and their particular capabilities is provided below.
The facility is extremely
well equipped for both analysis
and cell sorting. The Facility is available for use by outside groups on a special basis (please contact the Flow Staff for information as the rates will differ from those posted on the website). A brief description of the cytometers and their
particular capabilities is provided below.
Immunology Building 1st Floor
FACS Caliber HTS
Molecular and Experimetnal Medicine 2nd Floor
MEM FACS Caliber II
MEM LSR II
Science Park 3030 2nd Floor
FACS Caliber Science Park
- Digital LSR II -1, -2, -3, and -4. The most advanced benchtop analytical platform utilizes a number of technological advances: solid state diode lasers, advanced fluorescence detection with fiber optics and optimized beam
paths, and the digital signal processing used on the sorters. These advances combined together on an easy to use benchtop platform will give researchers the means to perform up to 10-color detection utilizing four
different excitation sources. The first of these instruments was purchased in 2003, a second in 2004, and a third in 2006 to accommodate increased user demand.
The LSR II-3 can be used with a 96-well plate loading format as well as standard tube loading. For more information, visit the LSR II webpage.
- FACS Canto. BD FACS Canto system is an easy-to-use benchtop analyzer that delivers proven performance, accuracy, and high-quality results. The BD FACSCanto II features many innovations including a true fixed
alignment flow cell to minimize startup time and improve reproducibility. The optical system features a patented design that maximizes signal detection and increases sensitivity and resolution for each color in a multicol
- FACS Calibur HTS. The institute has upgraded an investigator-operated instrument exclusively for analysis use. This instrument has been modified to optionally utilize a 96-well plate loading format as well as standard
tube loading. Additionally, this instrument uses CellQuestPro and PlateManager software designed for Mac OSX. In 2006, to accomdate the increased demand for basic 4-color flow applications,
a standard two-laser FACSCalibur was moved to Science Park 3030 Rm. 2030. This instrument is available to all users, but
is meant to assist those users located across the street from the Core Facility.
The detector arrangement and commonly used fluorescent probes
for the FACScan family of cytometers allow for a variety of multiparameter
The flow facility staff maintain
these analytical instruments in optimal working order and train
investigators in their use but do not operate the instruments for investigators.
The hourly charge for TSRI investigators is $55. User Calendars
are online and these instruments are available on a first
come first served basis. These instruments all have a built-in logiing software. Accounts are obtained
from the facility staff.
Digital LSR II -1, -2, -3, and -4. The most advanced benchtop analytical platform utilizes
a number of new technological advances: solid state diode lasers, advanced fluorescence
detection with fiber optics and optimized beam paths, and the digital signal processing
used on the DiVa sorters. These advances combined together on an easy to use benchtop platform
will give researchers the means to perform up to 10-color detection utilizing four different
excitation sources. For more information go to LSR II Webpage
The first of these instruments was purchased in 2003, a second in 2004, a third in 2006 and a fourth in 2010 to accomadate
increases user demand
Beckman-Coulter ViCell XR. This image based hemacytometer was purchased in June 2005 to facilitate
viability and cell counts for sorting experiments. Its ease of use has made it suitable as a user operated instrument
within the core. For more information go to ViCell XR Webpage
Immunology Building 1st Floor
FACS Vantage DiVa
FACS Aria I
FACS Aria II
MoFlo XDP I
MoFlo XDP II
- FACS Aria-I and -II Improvements in fluidics and optics have allowed for higher speed sorting (25,000 events/sec at 70psi using a 70um nozzle) with enhanced overall sensitivity.
The system uses fiber optic guided solid
state 488nm, 635nm, and 405nm diode lasers along with fiber guided emission collections (four colors plus scatter off the 488nm and two colors off the 635nm line and two colors off of the 405nm line).
These traits allow for an instrument that does not require the extensive alignment optimizations inherent to the jet-in-air sorters making for increased reproducibility in
identifying and sorting dim population.
For more information see the FACSAria Webpage.
- Propel Avalon . This new addition to the lab is the first of a new generation of low-cost sorters. It is designed to be used only with a 100um nozzle at lower pressures and has dual excitation lines (488nm and 568nm) and
four color detection making it ideal for basic sorting experiments using fluorescent reporter proteins and larger diameter cell lines.
- FACS Vantage DiVa I . TThis instrument is a veteran state of the art cell sorter. With three-laser capability, flexible optics and mixed gas tunable lasers, this instrument provides
flexibility in terms of applications. The Vantage has been upgraded with the Digital option (DiVa) in December 2002.
- MoFlo XDP-I and -II. TThis instrument has set the standard for cell sorting with functionality and speed. It is designed for researchers who desire high productivity. High viability, yield, and purity with an analysis rate of
100,000 events per second and sort rates of up to 40,000 events per second are standard. Each instrument has different possible fluorochrome combinations so see the laser line chart for more specific information or
contact the flow core staff.
optics as the third laser. A few examples are shown below:
It should be noted that the fluorochromes mentioned above are
the dyes most commonly available commercially. New dyes are continually
being developed and most of these can be used in this facility. Questions
regarding other applications should be directed to the facility staff.
Facility users are billed on an hourly basis
using the following scheme (outside users, please contact the Flow Staff for relevant rate information):
|Sorter Costs per Hour
|5 color + (staff operator)
|5 color + (user operator)
|Kinetics (Ca++) Analysis
All of the sorters (Vantage, FACStar Plus,
etc.) are operated solely by the trained flow facility staff members,
with the exception of some analytical experiments where experienced
users may run their own assay.
To reserve time on these instruments,
investigators should contact the flow facility staff at 784-8396 or 784-8286.
Users who book sorter time and determine that they need to cancel need to do so within 48 hours prior to their appointment or they will be charged a portion of their reserved time.
If cancellation is with 48 hours prior than there will be a 25% fee based on reserved time.
If they are 24 hours or less the fee will be 50% of reserved time. Outside entities will be charged full price based on their reserved times.
Data analysis is the responsibility of the
investigators. The flow facility makes available a variety of workstation
and software options for users. The user operated flow cytometers
all have Macintosh based computer systems for acquisition, and a Macintosh
G5 workstation with CellQuest, ModFit LT, WinList 4.0, FlowJo, and a variety
of other applications are available for data analysis and desktop
publishing. The current rate for the use of the workstation is $5.00/hr,
The technology of flow cytometry is rapidly
growing and highly technical. Errors in sample acquisition and/or
data analysis are common among poorly trained users and can lead to
misleading and/or unreliable data. The facility staff are committed
to providing training and education for all the facility users. Group training
sessions (at several levels) are available and individual help is
provided whenever possible. Contact staff personnel for specific information
regarding training sessions. Typically, new TSRI staff that will use
the flow cytometry facility are given an orientation (two 1.5 hour
sessions) to cover the broad scope of facility use and some specifics
on the use of the cytometers. Users that want additional training or people
new to flow cytometry are encouraged to contact the director for personal
assistance during several data aquisition and analysis sessions before
working extensively on their own. Contact the director, for any special
assistance or training at 4-8396 or firstname.lastname@example.org
Another area of focus also important for assuring
high quality work is the dissemination of reliable protocols for specific
flow cytometry applications. To this end, a protocol page is under
construction on the flow facility World Wide Web server for access
over the Internet.
Finally, one of the major long-term goals of
the facility is to collaborate with investigators in the development
of new, leading edge applications of flow technology. Investigators
are encouraged to discuss their ideas and/or needs with the director
of the facility.