FACSCalibur

The FACSCalibur has 4 fluorescence detectors plus forward- and side-scatter detectors.  See the table below to determine which fluorochromes may be used. 

Keep in mind that many fluorochromes will have overlapping emission spectra, and may be difficult to distinguish when used together.  Be sure to check the excitation and emission spectra of the fluorochromes you plan to use to optimize your experiment.  The BD Biosciences Fluorescence Spectrum Viewer may help you determine whether the fluorochromes you want to use will have a significant overlap.  We also have a list of some fluorochromes, dyes and fluorescent proteins you may check. 

Analysis

The FACSCalibur computer uses CellQuest software to collect and analyze data.  Using CellQuest, data may be analyzed on dot plots and histograms with various gates.

Your data will be analyzed as part of your flow cytometry appointment as long as time allows, or you may set an appointment specifically for data analysis.  Because analysis requires time on the cytometer computer, analysis-only appointments are charged the same rate as data acquisition/analysis.  You may choose to work with facility personnel to analyze your data, or you may analyze data on your own. 

WinMDI is a free program available for use on PC's.  There is also a tutorial for the program to help you learn to use it. 

Other programs are also available under our useful links menu. 

Data Delivery

The FACSCalibur is connected to the UAMS network.  Data from the Calibur may be stored temporarily on the network at \\resnet\FACS in a designated folder.  Otherwise, data may be stored on CD or Zip disk directly from the FACSCalibur computer.

Sorting with the FACSCalibur

*** The FACSCalibur will only be used for sorting if there are problems with the FACSAria.  The FACSAria is primarily a cell sorter, and the FACSCalibur is primarily for analysis only .***

Sort Capability:  Capture rate is 300 cells/sec; aerosol-free; cells sorted into one 50 ml conical tube at a time (10 min per tube).

The maximum sort rate on the FACSCalibur occurs when the event rate (#cells/sec) is 2000.  To achieve this, cell concentration should be approximately 10 7 cells/ml.  Higher or lower event rates result in lower sort rates.  This will not affect the amount of time required to sort, but will affect the cell recovery.  Cells sorted by the FACSCalibur are diluted into 50 ml conical tubes.  Sorting buffer drips at a constant rate into the collection tube, whether cells of interest are detected and sorted or not .  A higher sort rate will ensure a higher number of sorted cells. 

Samples must be provided in a 12 x 75 mm polystyrene or polypropylene tube (Fisher brand # 14-9563A, 14-9561D or equivalent).