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ARL/AZCC Flow Cytometry Shared Service
Reminder: SWEHSC Investigators (including their staff and students) should contact the Cellular Imaging Shared Service directly for assistance.
While regular users of this facility can learn how to operate the FACScan, this is primarily a full-service lab. Typically users bring their samples to the facility and the lab staff scan or sort the cells as needed. Service is by appointment to ensure that time sensitive experiments have access to the facility at the appropriate time points. The lab staff are available to assist users with software analysis of their data.
Users should contact the facility for sample preparation advice and/or protocols.
The BD Biosciences FACSAria cell sorter has three lasers (407/488/633 nm) and can detect eleven colors. It is very fast, being able to count over 30,000 events/second and sort 20,000 cells/second. It can sort four different populations of cells onto tubes of many sizes, plates, slides and culture dishes. The instrument can sort out a single cell if needed.
The BD FACScan is a flow cytometer that can be used for more routine assays. It does not perform sorting and has a smaller number of parameters that it can measure.
The following list of includes some of the measurable parameters of cells and particles using flow cytometry (list courtesy of the FACS facility) :
- Cell size/shape
- Cytoplasmic granularity
- Cell surface antigens
- Membrane integrity (viability)
- DNA content, and base ratio
- DNA degradation (apoptosis)
- Chromatin structure
- DNA synthesis
- Membrane permeability
- Membrane potential (dye/drug uptake)
- Membrane-bound/Cytoplasmic Calcium
Advantages: An ideal technique for performing quantitative fluorescence measurements on a population of cells. Data can be acquired quickly, many parameters can be measured simultaneously and the cells of interest can be recovered by sorting out specific sub-populations. Other types of studies available include kinetics and FRET.
Disadvantages: Data is of a population of cells, users will not know which individual cell produced a particular measurement. While sub-populations of cells can be recovered, frequently the sample is analyzed and then discarded. This technique measures cells, therefore intact tissues usually require pre-treatment with a enzyme to release the individual cells for analysis.
