UCR

Institute for Integrative Genome Biology



FACSAria


Genomics Services

FACSAriaThe Flow Cytometry Facility operates a BD Biosciences FACSAria, which is a high-speed cell sorter with 3 lasers giving it the ability to test 9 colors per tube. The FACSAria is equipped with BDTM Automated Cell Deposition Unit (ACDU) and a BDTM Aerosol Management Option.

FACSAria

The FASAria instrument is designed to sort cells in liquid by passing them through a narrow orifice where their fluorescent properties (typically from dyes, expression of fluorescent proteins or immunofluorescence) can be detected. Cells can then be sorted physically. The FACSAria is operated by a Genomics Core staff member, and in general, is not operated by customers except by arrangement. We offer a service utilizing the instrument which is billed on a per hour basis. In addition, we offer training in flow cytometry as well as advice on experimental design. For details, contact our staff.

Protocols

Cell Samples

First-time users are strongly encouraged to discuss experimental objectives with Core staff if they are contemplating this approach. Samples that have been sorted successfully include mammalian and plants cells.   A critical factor in successful cell sorting is cell diameter. The FACSAria has a nozzle diameter about 100 micrometers. As a general rule, the cell diameter should not exceed one-fifth of the nozzle diameter. This ensures the stability of the stream during the sort. Larger diameter particles affect the droplet breakoff or may even clog the nozzle. Another important factor in successful sorting is cell morphology that deviates from a spherical shape. Irregular shaped cells may require greater nozzle diameter. These factors should be discussed with our staff prior to any experiments.

Buffer Selection

The proper design of sorting buffers for both the pre-sort sample and your collected sample is crucial for success. The following is a basic recipe for mammalian cells and some suggestions for modifications that might be relevant to your particular experiment. Culture media is not an ideal sort buffer for two reasons: the pH regulation fails under normal atmosphere causing the media to become basic and the calcium chloride in most culture media is not compatible with the phosphate component of the instrument sheath buffer (the Basic Sorting Buffer without additional protein). This results in precipitation of calcium phosphate. Following the suggested recipes below will help maximize the recovery and viability of your sorted cells.

Basic Sorting Buffer

1x Phosphate Buffered Saline (Ca2+/Mg2+ free)
1mM EDTA
25mM HEPES, pH 7.0
1% Fetal Bovine Serum (heat-inactivated)

0.2 micrometers filter sterilize, store a 4oC

For Clean Lymphoid Cells: The buffer can be simplified to HBSS with 1% FBS. The additional cations in the recipe promote better viability. Since these cells are not prone to clump, the lack of EDTA is not a problem.

For Sticky Cells: Raise the concentration of the EDTA to 5mM and use FBS that has been dialyzed against Ca2+/Mg2+ free PBS. Some activated cells become clumpy and the chelators (EDTA) will help reduce cation-dependent cell-to-cell adhesion.

For Adherent Cells: In order achieve good single cell preparations, one must start at the moment of detaching your cells from the plate. Typically, the trypsin (or other detachment buffer) is quenched with culture media or a PBS/FBS buffer. This is problematic because it reintroduces the cations that facilitate the cells reattaching to the plate (or each other). One must use a cation-free FBS buffer in order to stop the detachment. Additionally, the level of EDTA can be increased if necessary (but too much EDTA can be deleterious).

For Samples with a High Percentage of Dead Cells: If there are a large number of dead cells in the prep, it is likely that there is soluble DNA from the dead cells that will come out of solution. This DNA will start to coat the cells and will lead to severe clumping. The addition of 10U/mL DNAase II to the buffer recipe will help reduce DNA associated clumpiness.

Cell Concentrations

Guidelines concerning the correct concentrations of cells of sorting.

Selecting Reagents for Multicolor Flow Cytometry

Guidelines concerning dyes for multicolor sorting.

 For additional information, please view the Flow Cytometry and Sorting Presentation

Helpful Links:

 

The Flow Cytometry Facility in the Institute for Integrative Genome Biology (IIGB) at the University of California, Riverside has two BD Biosciences cytometers. The FACSAria is a high speed cell sorter with 3 lasers giving it the ability to test 9 colors per tube. The FACSAria is equipped with BDTM Automated Cell Deposition Unit (ACDU) and a BDTM Aerosol Management Option.

The FACScan is a one laser 3 color cytometer. The Center is staffed by a dedicated flow cytometer operator. The rate for the FACSAria run by a staff operator is $80 per hour and for the FACScan run by the client is $20 per hour.


More Information

General Campus Information

University of California, Riverside
900 University Ave.
Riverside, CA 92521
Tel: (951) 827-1012

Career OpportunitiesUCR Libraries
Campus StatusDirections to UCR

Genomics Information

Institute of Integrative Genome Biology
2150 Batchelor Hall

Tel: (951) 827-7177
E-mail: Aurelia Espinoza, Managing Director

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