In addition to the most powerful cell sorter BD FACSAria III, the Cell Analysis & Immunology Core Facility owns advanced analyzers such as BD LSRII and BD FACSArray. The Core is able to sort human as well as animal cells under aseptic condition at high speed with high purity and yield. BD LSRII is suitable for (not limited to) immunophenotyping, intracellular cytokine and signaling pathway study, cell cycle determination and apoptosis detection by several different ways. BD FASArray provides an easy, fast way to detect cell-secreting proteins or intracellular proteins with high-sensitivity without limitation of sample volumes. Additional capabilities include an Auto MACS cell sorter, a BioRad Bio-plex system, an AID Elispot reader and a Luminoscan. The auto MACS separator is a computer controlled magnetic cell sorter for isolation of virtually any cell type. The system is compatible with all MACS cell separation regents. There are several separation programs available allowing different cell selection strategies. Employing the MACS magnetic cell separation technology the auto MACS separator is capable of sorting more than 10 million cells per second from samples of up to 109 total cells. The BioRad Bioplex system is a fully intergrated system combining hardware, Bio-Plex Manager software, system validation and calibration tools, assays, and beads. The system ensures accurate and reproducible results. The AID Elispot Reader System is a combination of classic AID reader unit and the AID EliSpot software to acquire, analyze, and quantify the spots according to the user’s settings. The Luminoscan Ascent is a sensitive and flexible microplate luminometer. Combined with the powerful Ascent software, it handles both glow and flash type reactions. It offers great advantages for different types of assays. These include reporter gene assays, immunoassays with luminescent substrates, cytotoxicity and cell proliferation assays, Intracellular Ca+2 assays, ATP assays, DNA quantification.
In addition to instrumentation, the Facility intends to collaborate with the investigators and contribute to all aspects of the research process. This includes consultation in experimental design, technical assistance in the operation of instruments, trouble shooting, data analysis and storage, interpretation of results and preparation and production of presentation graphics. The Facility is also communicating with investigators in other institutions and is devoted to introducing and developing new applications on our flow cytometers. In this way, investigators are provided with unlimited possibilities to answer questions regarding nature and function of cells by flow cytometry.
The Cell Analysis & Immunology Core is housed within the LCRC and offers a wide-array of flow cytometry services for LCRC researchers. In addition to flow services, this location is set up with an Auto MACS, BioRad Bioplex System, AID Elispot Reader System, and Luminoskan Ascent.
Equipment, Software, Resources
BD LSRII analyzer
A three-laser bench-top flow cytometer designed with all-digital signal collection and processing and a fixed alignment fiber optical system engineered to achieve the greatest amount of signal detection for each laser. The standard optical filters on the LSR allow the detection of (but are not limited to) the following fluorophores: PE-Cy7, PE-Cy5.5, PE-TexasRed, PerCP, PerCP-Cy5.5 PE, FITC, PI, APC-Cy7, APC, Hoechst, DAPI, Cascade Blue, Pacific Blue, Alexa Fluor 405 and Alexa Fluor 430. It can simultaneously detect 10 parameters (2 scatter and 8 fluorochromes). Additionally, the LSU and Tulane sites both have an HTP attachment which allows high through put analysis of 96 well plates.
BD FACSAria III
The newest cell sorter which is equipped with six solid state lasers. The computer is capable of collecting 20 parameters simultaneously (2 scatter signals and 18 fluorescent parameters). It is designed to be a high precision sorter, which allows for acquisition rates of up to 70.000 cells per second and sorting rates up to 70,000 cells per second while maintaining high purity and cell recovery yields. Cells can be collected under non-aseptic or aseptic condition. An automated cell delivery system (ACDU) permits sorting individual cells for direct cloning into 384 well plates.
A compact system rapidly detects and quantifies concentrations of secreted proteins, proteins in cell lysates, and cell-associated proteins using small sample volumes. It can be used to detect apoptosis, cytokine and chemokine profiling and phosphorylation of key signal transduction proteins by multiplexed BD cytometric bead array (CBA) products. FACSArray is a very flexible system suitable for many different types of application and this system is fast and requires minimal training.
The BD FACSCalibur system combines unique dual-laser technology. Designed specifically to support a wide range of applications, the BD FACSCalibur system is a fully integrated multiparameter system that offers investigators the performance and flexibility required for a variety of research applications.
The auto MACS separator is a computer controlled magnetic cell sorter for isolation of virtually any cell type. The system is compatible with all MACS cell separation regents. There are several separation programs available allowing different cell selection strategies. Employing the MACS magnetic cell separation technology the auto MACS separator is capable of sorting more than 10 million cells per second from samples of up to 109 total cells.
BioRad Bioplex System
This is a fully integrated system combining hardware, Bio-Plex Manager software, system validation and calibration tools, assays, and beads. The system ensures accurate and reproducible results.
AID Elispot Reader System
It is a combination of classic AID reader unit and the AID EliSpot software to acquire, analyze, and quantify the spots according to the user’s settings.
- Analysis and interpretation: The facility will assist with the retrieval and analysis of data and interpretation of results. There are several packages of software available to analyze list data, including Cell Quest, FlowJo, Microsoft Word, Excel, PowerPoint, as well as Photoshop.
- Data archival: The facility adheres to a file saving structure based on the investigators email/logon ID. Each user will obtain a logon ID and a password (usually their last name) for access to the instruments. The investigators need to create a folder with his last name. This is where the data will be saved. The FCS files will be named as mmddyyxxxF, where m=month, d=day, y=year, xxx=users’ last name, F= name for instrument used in the experiment. Data is backed up at the end of each day to an archival computer. At the Tulane site, investigators can access this computer over the network to retrieve data for approximately 6 months. At LSU investigators will need a flash drive or CD to retrieve data, as no network access is available. All data is permanently archived to CD-ROM storage disks.
All instruments are cleaned and quality control tested daily to ensure the highest level of reproducibility of data.
- Sorting: The sorter BD FACS Aria III is run exclusively by the operator and highly trained authorized users. The fluidic systems are cleaned and sterilized with antiseptic detergent to maintain aseptic conditions and eliminate crossover contamination between samples.
- Analysis: An operator is available for running samples on BD LSRII and FACS Array. The facility can also assist investigators with the setup and running of samples. Investigators are encouraged to learn how to run their experiments on the LSRII and FACS Array to enable independent usage of the facility allowing for the flexibility to schedule time for analysis during the evenings and weekends. For this purpose, users need to be trained and demonstrate that they know the proper set up, running and shut down procedures.
Among the available analyses:
- immunophenotyping (cell surface antigens and receptors)
- intracellular proteins, cytokines, calcium flux, pH and free fatty acid,
- signal transduction pathways,
- DNA and RNA content, cell cycle, DNA synthesis,
- apoptosis (programmed cell death) analysis, based on:
mitochondrial membrane potential
down regulation of BCL-2
activation of caspases (cysteine proteases)
cleavage of the DNA repair enzyme PARP
DNA fragmentation (TUNEL Assay with anti-BrdU labeling)
translocation of phosphatidylserine from the inner cytoplasmic membrane to the cell surface (Annexin-FITC/PI)
The Cell Analysis & Immunology Core Facility is currently supported by LCRC Funds. A chargeback system will be implemented whereby grants will be charged for investigator services. Revenue generated from the charges will cover a portion of the operational expenses through cost recovery.
(50% discount for LCRC Program Members 30% discount for LCRC Contributing Members 10% discount for LCRC Associate Members 0% discount for non-members)
Augusto Ochoa, MD
Dorota Wyczechowska, PhD – LSU
Mary Price, BS – Tulane