ARL/UACC Flow Cytometry Shared Service

 

Location: University of Arizona Cancer Center, room 0935
Contact: Paula Campbell, 621-2047
Administration: AZ Research Labs, Division of Biotechnology and University of Arizona Cancer Center.
Fees: see the facility's online list of service fees
WWW: http://cytometry.arl.arizona.edu/index.php

Reminder: SWEHSC Investigators (including their staff and students) should contact the Cellular Imaging Facility Core directly for assistance.

 

Full service:

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.

 

Instrument Capabilities:

Full service only:

The BD Biosciences FACSAria III cell sorter has five lasers (355/405/488/561/633 nm) and has seventeen fluorescence detectors. 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.

User-operated or service work (upon request):

The BD FACSCanto II is a flow cytometer that can be used for more routine assays. It does not perform sorting and has a smaller number of parameters ( 2 lasers, 6 fluorescence channels and 2 channels for Forward Scatter and Side Scatter) that it can measure.

The LSR II (located in MRB) is equipped with 5 air-cooled lasers, 16 fluorescent channels and 2 channels for Forward Scatter and Side Scatter. It is a state of the art digital flow cytometer, containing octagon and trigon optical arrays, allowing increased signal sensitivity.

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 and Disadvantages:

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.