Equipment

The Cancer Center Flow Cytometry Core Facility is equipped with 5 flow cytometers that are categorized into 3 types:

• 3 traditional analyzer flow cytometers that are typically operated by investigators themselves at the BSL1 level.
• A state-of-the-art imaging flow cytometer that is typically operated by investigators themselves at the BSL1 level.
• A cell sorter flow cytometer that is typically provided as a service to investigators, which can operate at the BSL2+ level.

In order to select the best fluorophores for use in a specific application on a particular flow cytometer, it is necessary to know the laser configuration of the cytometer and its optical configuration and detectors. The facility's lasers are summarized in the following table. Each cytometer's configuration is detailed in the section specific to the cytometer, in the sections that follow.

Analyzer Flow Cytometers

The three traditional bench top analyzer flow cytometers in the lab are manufactured by Becton Dickinson Immunocytometry Systems, San Jose, Calif., and consist of a two-laser FACSCalibur, a two-laser FACSCanto and a three-laser LSRII. Know that FACS™ is an acronym for Fluorescence-Activated Cell Sorting and is Becton Dickinson's registered trademark.

Becton Dickinson FACSCalibur analog analyzer flow cytometer [four-color, two-laser system]

The core is equipped with a two-laser FACSCalibur flow cytometer that is equipped with a low-power (15mW) blue laser that emits light at 488 nanometers as well as a low-power (30mW) red laser (diode) that emits  light at 633 nanometers. The FACSCalibur has two detectors for laser-light scatter and four detectors for fluorescence in the green, orange and red/dark red regions of the color spectrum. The detector array permits the use of a great number of fluorophores, allowing for up to seven parameters for every particle interrogated (time is also a parameter). The optical filters on the FACSCalibur are not interchangeable (each fluorescence detector "sees" on a single fluorescence color) so the choices of fluorophores are similarly constrained, however, a tremendous number of fluorophores can be detected with its configuration. The FACSCalibur is equipped with pulse processing for cell doublet discrimination.

 

Becton Dickinson FACSCanto digital analyzer flow cytometer [seven-color, two-laser system]

The FACSCanto has optical detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on the FACSCalibur. The FACSCanto is also equipped with pulse processing for cell doublet discrimination and can provide investigators with up to 10 parameters for every particle interrogated. The FACSCAnto has seven detectors for fluorescence in the following configuration:

 

  Becton Dickinson LSR II digital analyzer flow cytometer [eight-color, four-laser system]

Similar to the FACSCanto, the LSR II has optical detectors and removable optical filters that can permit the detection of different fluorophores and fluorophore combinations than are regularly used on a FACSCalibur, however, the lab’s LSR II has eight, not seven, detectors for fluorescence and four, not two, lasers in the configuration as shown below. The LSR II is also equipped with pulse processing for cell doublet discrimination and provides investigators with up to 11 parameters for every particle interrogated.

 

 

 

 

Imaging Flow Cytometer

The state-of-the-art imaging flow cytometer in the lab is manufactured by Amnis of Seattle, Washington.

Amnis ImageStream^X digital imaging flow cytometer [6 channels, 4 lasers].

The ImageStream^X, the first in the state of Georgia, provides a breakthrough in flow cytometry by providing imaging capability to flow cytometry (multispectral imaging flow cytometry). This unique technology affords identification of a single cell based on fluorescence dye intensity, morphology of the cell by bright field imaging, as well as morphology and distribution of fluorescence markers (reporters). Visit the manufacturer’s website for additional information. Our ImageStream^X is equipped with most available options, i.e., Enhanced Depth of Field, Multimag (20x or 60x in addition to standard 40x magnification) and Full Brightfield as well as 4 lasers (blue, red, violet and yellow).

 

 

 

ImageStream^X Frequently Asked Question

ImageStream^X Sample Preparation Guide

ImageStream^X Experimental Design Worksheet

ImageStream^X Data Acquistion Form

ImageStream^X Brochure

INSPIRE User Guide

IDEAS User Guide

   

 

Cell Sorter Flow Cytometer

The cell sorter flow cytometer in the lab is manufactured by Becton Dickinson Immunocytometry Systems, San Jose, Calif., and consists of a two-laser FACSAria IIu.

Becton Dickinson FACSAria IIu cell sorter flow cytometer [9 color, 2-laser system with optional 4-way sorting, aerosol containment, cloning and temperature control system].

The FACSAria IIu is equipped with 9 fluorescent detectors and two lasers as detailed in the following table. Its fluidics system has been upgraded from its predecessor, a FACSAria I, hence the FACSAria IIu designation. The FACSAria IIu is housed inside a negative-pressure containment suite and is placed inside a Baker BioProtect III biosafety cabinet for safety of operation. It is capable of accommodating sorting at the BSL2+ level.

The FACSAria, because it is a closed-cuvette sorter, has sensitivity equal to that observed in the analyzer flow cytometers.

The FACSAria can physically sort up to four populations concurrently and it is able to sort them into a great variety of collection devices:

• Microscope slides 

• Microtubes 

• 12 x 75 mm test tubes 

• 15 conical tubes 

• 50 conical tubes 

• Multiwell plates (24-, 96-, or 384-well plates, etc.; 60- or 72-well Terasaki, etc.)

 

Support Equipment

Automated cell counter

    Nexcelom Cellometer Auto T4 uses 20µl of sample with disposable counting slides and performs:

•  

   • Cell concentration
   • Imaging
   • Cell size histograms
   • Viability

The Auto T4 accommodates cells that are in the size range of 5-60 microns that are in the concentration  range of 0.25 - 10.0 x 10⁶.

Tutorials for using the Nexcelom Cellometer Auto T4..

 

 

Computer analysis workstations

There are 2 computer analysis workstations available that facilitate the analysis, presentation and publication of flow cytometry-generated data.

A twin 2.0 GHz dual-core Intel Xeon Mac Pro running OS X Lion (10.7) and equipped with:

• FlowJo v9 
• ModFit LT 3.3
• Photoshop CS5
• Illustrator CS5
• Office for Mac 2011

Tutorials for learning how to use FlowJo are available and can also be accessed in the Protocols & Useful Links section of this Web site.

                                                                   A dual Intel Xeon 2.26GHz with 2 quad-cores on each processor (yes, that’s 16 processors) Dell Precision T7500 running Windows 7 64-bit with 4 GB memory and equipped with:

• IDEAS v5 (for ImageStream× data file analysis)
• Photoshop CS5
• Illustrator CS5
• Office 2010

 

 

 

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