Mission
The objectives of the CFAR Flow Cytometry Core Facility are:
1) Service: To provide cell sorting and analytical flow cytometry services.
2) Education and Training: To provide up-to-date information regarding the use of flow cytometry in research applications.
3) Innovation: To ensure that users can take advantage of current technological advances and employ state of the art techniques in their research activities.

Facility Description
The CFAR Flow Cytometry Core has been in operation since 1989. Marion Spell, the Core Manager, has been manager of the facility since its inception and is highly experienced at operating the instruments. Marion Spell and the Core Director, Allan Zajac, provide both new and established users with help and advice regarding the use of the facility, and the design and interpretation of experiments which utilize flow cytometry. The Core is located in the Lyons Harrison Research Building (Room 610 of the CFAR BL-3 facility for sorting and Room 624 for analytical cytometry).

Flow Cytometers. The Core is equipped with a high speed BD FACSAria Cell Sorter and a BD LSRII Cell analytical flow cytometer.

FACSAria. The BD FACSAria Cell sorter is used for analytical and preparative flow cytometry. The FACSAria design allows for acquisition rates of up to 70,000 cells per second and sorting rates up to 40,000 cells per second while maintaining high purity and cell recovery yields. It can simultaneously detect up to 15 parameters (2 scatter signals and up to 13 fluorescent parameters) by utilizing 3 solid state lasers (laser outputs at 488nm, 633nm, and 407nm) and a fixed optical alignment system allowing for multiple configurations and a wide range of fluorochromes (see section below). The FACSAria can sort 1, 2, 3, or 4 populations simultaneously and is equipped with an Automated Cell Deposition Unit for sorting into multiwell plates or slides. The FACSAria is equipped with an Aerosol Management System that evacuates any aerosols from the sort chamber providing an additional level of safety for the operator and has a refrigeration system that allows cooling of the sort sample as well as the sort collection tubes. It can accept various sample input tubes including 1 mL microtubes, 12 x 75-mm test tubes, and 15 mL tubes into the sample and collection chambers. It is equipped with a Hewlett Packard XW4000 Computer using Microsoft Windows NT2000 operating system. Data can be stored or transferred using USB Flash Drives or the internal 4.7GB DVD+RW/CD-RW. The FACSAria is also equipped with BD FACSDiva software that is used for the collection, storage, and analysis of the digital data.

BD LSRII. The BD LSRII Flow Cytometer, used for analytical flow cytometry, is designed with all digital signal collection and processing that incorporates 4 solid state laser beams (laser outputs at 488nm, 633nm, 407nm, and 355nm) and a fixed alignment fiber optic optical system engineered to achieve the greatest amount of signal detection for each laser. It can simultaneously detect up to 20 parameters (2 scatter and 18 fluorescent parameters) and the optical system can be setup in many configurations allowing the use of a wide range of fluorochromes (see section below). The BD LSRII is equipped with a Hewlett Packard XW4000 Computer using Microsoft Windows NT2000 operating system. Data can be stored or transferred using the USB Flash Drives or the internal 4.7GB DVD+RW/CD-RW. It is also equipped with BD FACSDiva software that is used for the collection, storage, and analysis of the digital data.

Standard Optical Configuration of Instruments-(Other configurations are available)

Analysis Workstations/Software. The flow facility has two off-line analysis workstations available to users that can be utilized for analysis of the digital data. A G4 MacIntosh workstation (OSX) with the FlowJo cytometry software package is available for Mac users and a Dell workstation (Windows XP) with the BD FACSDiva cytometry software package is available for Windows users. The FlowJo and FACSDiva programs are designed specifically to analyze the digital data collected on the LSRII and FACSAria while giving the user a wide range of options for data analysis and graphics presentation. The Core also has other software packages available which include CellQuest, CellQuest Pro, CellQuest Pro for OSX, ModFit LT, WinMDI (PC version), and Winlist 3.2 (Mac version). The Core also recommends the FCS Express software packages for Windows PC operating systems.

Research Information
Types of Fee-Based Services Offered. The following types of fee-based services are offered by the Core. The level of assistance provided is dependent upon the knowledge level and experience of the Core user.

• Operator assisted use of the flow cytometers
• Operator unassisted use of the flow cytometers for supported assays (requires prior authorization by flow facility personnel)
• Offline operator software analysis of flow cytometric data
• Education services are offered to users of Flow Cytometry Core and are tailored to the needs and knowledge level of the user
• Consultation service is offered as a free service for all users of flow cytometry
  

Flow cytometry provides an exceptionally powerful tool for analyzing and isolating populations of cells. The types of analyses performed are determined by the needs of the researchers; researchers often benefit from prior consultation with facility personnel to ensure the service provided is appropriate for the specific research application. The publication, "Current Protocols in Cytometry" is available in the facility as a reference for development of assays and to provide protocols for utilizing flow cytometry in a wide range of research applications. The following are a few examples of the numerous assays that can be performed by the CFAR Flow Cytometry Facility: immunophenotyping with up to 18 fluorochromes; quantitative fluorescent intensity assessment; analysis of HIV proteins expressed on the cell surface; intracellular immunofluorescence, including intracellular cytokine analysis; cell sorting, including the sterile sorting of HIV infected cells; single cell cloning; analysis of apoptosis and viability assays; analysis of GFP expression; analysis of cell cycle (G0/G1, S, G2 + M); intracellular pH/Calcium; mitochondrial function assays; measurement of cytokines; and cytometric bead array assays (CBA).

Sample Preparation
Analytical. Samples should be prepared in the investigator's lab and must be in suitable condition for loading onto the cytometers. Additionally, the samples should be in BD Falcon 12x75mm polystyrene tubes at maximum concentration of 1x106 cells/ml. The concentration may be increased as needed for the type of assay. In these instances the user should consult with the Core manager.

Sorting. For sorting, cell concentrations can range from 3 x106 up to 15 x106 cells/ml in a variety of tubes. Cell preparations with aggregates may not flow evenly through the 70 um nozzle typically used on the FACSAria sorter and all samples must be filtered prior to sorting. The aggregates can be removed by filtration using commercially available filters which include the 70um FALCON cell strainer (35-2350) or 35um FALCON strainer cap/ test tube (35-2235).

Controls. Investigators should always include a negative control such as an unstained sample or a sample stained only with the secondary reagent. In multicolor analyses, single stained control samples for each of the fluorochromes used should be included to facilitate electronic compensation of the spectral overlap between fluorochromes. The Mario Roederer web site provides a discussion of the necessary considerations for appropriate compensation.

Scheduling
Sessions can be scheduled by contacting Marion Spell in person, via phone, or via e-mail. Reservations should be made at least 24 hours in advance and will require your name, phone number, the instrument that will be used, the time you wish to reserve, an Oracle account number for charging purposes, and whether a FACS/facility operator will be needed. Notice of cancellation of an appointment must be given at least 24 hours in advance in order to avoid being billed for the scheduled time.

Contact Information
Core Director: Allan Zajac, PhD
Email: azajac@uab.edu
Phone: 205-975-5644

Core Manager: Marion Spell
Email: mlspell@uab.edu
Phone: 205-975-3227

Web Site: http://www.uabcfar.uab.edu/cores/flowcytometry

Approved by: Marion Spell, Core Manager
Date: February 21, 2007

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