Moody Foundation Flow Cytometry

About

Frances Moody-Dahlberg visits the CRI’s Flow Cytometry Facility

The Moody Foundation Flow Cytometry Facility, located within CRI, is a shared resource laboratory (SRL) committed to providing cutting-edge flow cytometry services to scientists at CRI, UT Southwestern and other institutes. The facility is named after the Moody family, who donated $2.5 million to renovate, staff and buy equipment for CRI’s Flow Cytometry Facility in 2013.

The facility offers state-of-the-art analytical cytometry and high-speed sorting either in an investigator-operated format or facility staff assisted format. Our instrumentation is compatible with a wide variety of flow cytometry applications. We also offer training for research personnel interested in learning how to design flow cytometry experiments, operate the instrumentation and analyze data.

  • We provide several core services, including:
  • Experimental design
  • Protocol development
  • Sample staining
  • Instrument operation
  • Data analysis
  • Graphical production
  • Sample Processing as a Service (SpaaS)

The CRI flow cytometry shared facility has three cells sorters, two FACS Aria II SORP and one FACS Aria Fusion SORP, all in a biosafety hood for sorting primary human cells. We also have two cells analyzers. One is a special order FACS LSRFortessa SORP with High Throughput Sampler (plate loader) that provides fully automated and rapid sample acquisition. This system also includes an enhanced dual FSC detection system (PMT + diode) with the ability to detect 0.2 um particles from noise. The other is a special order FACS Canto RUO – 12 parameters – with tubes loader.

If you would like to gain access to the facility, please visit CRI PPMS, and complete an account creation request. For answers to commonly asked questions and helpful links, please refer to our FAQ.

Introduction to Flow Cytometry

Flow cytometry is a powerful tool for investigating many aspects of cell biology due to its ability to quantitatively analyze individual cells and identify different subpopulations of cells. This makes flow cytometry a valuable tool for cell analysis and cell sorting. Cell features are detected primarily by fluorescence though significant information, such as cell “size” and “granularity”, is also contained in the way light is scattered by the cells. The power of single cell analysis is heightened by the ability to analyze multiple parameters simultaneously. Flow cytometers equipped with the latest technology available, such as those offered in our facility, can also analyze and sort cells at extremely high rates. Applications of flow cytometry include immunophenotyping (using fluorescently labeled antibodies or ligands), measurement of DNA and RNA content for cell cycle analysis, apoptosis, and cell viability. These techniques can be used to answer a diverse range of experimental questions.

Flow cytometry is increasingly being used for preparation of gene libraries by sorting green fluorescent protein (GFP)-marked E. coli transfectants that are directly cloned following sorting. Although flow cytometry is mostly applied to the analysis of cells, it is applicable to any resolvable particle such as chromosomes or bacteria.

Facility Access

The Moody Foundation Flow Cytometry Facility is open to all scientists at CRI, UT Southwestern and other interested institutions.

For untrained users, our staff is available to operate instrumentation Monday through Friday from 9 a.m. to 6 p.m. Facility training courses are available for those interested in being self-operators through flow facility staff members. Training includes mastery of sample preparation, instrumentation and analysis software. Flow cytometers are relative measurement devices, and, therefore, outcomes depend on proper setup and use.

For qualified self-operators, the Facility is open 24/7.

Instrumentation

Our facility is equipped with three cell sorters and two cell analyzers.

Two cells analyzers: one special order FACS LSRFortessa SORP with High Throughput Sampler (plate loader) that provides fully automated and rapid sample acquisition and one special order FACS Canto RUO – 12 parameters – with tubes loader. These instruments operate on a PC-based platform using FACSDiva (version 8) software. To schedule time with our instruments, please visit CRI PPMS.

We offer three cells sorters, two FACS Aria II SORP and one FACS Aria Fusion SORP, all in a biosafety hood for sorting primary human cells. For help getting great data, please review our tip sheet.

Analyzers

FACS Canto RUO

The FACSCanto RUO is equipped with three lasers (405 nm, 488 nm and 633 nm) that offer simultaneous 12-parameter detection and a FACSLoader. This state-of-the-art instrument offers fixed optical alignment as well as automated compensation and housekeeping routines, making it highly desirable to operate.

FACS LSRFortessa SORP

The FACS LSRFortessa SORP is equipped with four lasers (405 nm, 488 nm, 561 nm and 633 nm) that offer simultaneous 18-parameter detection and a High Throughput Sampler (HTS) that provides fully automated and rapid sample acquisition. The system also includes an enhanced dual FSC detection system (PMT + diode) with the ability to detect 0.2 µm particles from noise.

This state-of-the-art instrument offers high power lasers that improve the detection of rare events as well as automated compensation and housekeeping routines, making it highly desirable to operate.

Cells Sorters

FACS Aria II SORP (4 lasers and 5 lasers) / FACS Aria Fusion SOP

The FACSAria II SORP and FACS Aria Fusion SORP offer state-of-the-art features in the most advanced cell sorter on the market. BD FACSAria II SORP and FACS Aria Fusion SORP have air-cooled lasers and fixed-alignment cuvette flow cells, which allow achieving superior fluorescence sensitivity. BD FACSAria delivers high-speed multicolor sorting with digital acquisition rates of up to 20,000 events/second. It is able to achieve sort into two- and four-way bulk sorting devices for a variety of tube sizes and can sort using Automated Cell Deposition Unit (ACDU) to multiwell plates or microscope slides.

Quick reference guides are available for the FACSAria II SORP and FACSAria Fusion SORP. Please review our guidelines for help with sample preparation for sorting.

Two FACSAria II SORP are available in the facility.

Four-laser system (405 nm, 488 nm, 561 nm, 633 nm), which simultaneously collects multiple emission wavelengths (up to 13 fluorescents detectors):

Five-laser system (305 nm, 405 nm, 488 nm, 561 nm, 633 nm) that accommodate up to 17 parameters – 15 colors and 2 scatters:

Also available is one FACSAria Fusion SORP, which is a give-laser system (305 nm, 405 nm, 488 nm, 561 nm, 633 nm) that accommodate up to 20 parameters – 18 colors and 2 scatters. The three FACSAria (II and Fusion) are in class II biosafety cabinets designed to provide personnel, product and environmental protection from potentially hazardous, aerosolized particulates.


Confocal Microscopy

The Leica TCS SP8 has been designed for confocal microscopy with optimal photon efficiency and high speed. All optical components are matched toward preserving fluorescence photons for image contrast and to improve cell viability in live cell imaging. Backing up this sensitive detection are a high speed scanning system with up to 428 frames per second, large field of view of field number 22 and accelerated Z-stacking by a novel mode for the SuperZ galvanometer called Galvoflow. This suits the instrument for the most demanding samples, highest speed while offering full confocal resolution.

Flow Cytometry Tools

FlowJo

The Flow Cytometry Facility administers the UTSW site license for FlowJo analysis software, which allows us to offer a heavily discounted version of the software for our users. To participate in this program, you must be affiliated with UT Southwestern and be able to provide a valid account number for this license for billing. To obtain a serial number for your computer complete this form and email it to nicolas.loof@utsouthwestern.edu.

Our flow cytometry experts are available to help analyze data but you can also find videos and tutorials with demo data on the FlowJo website.

Spectral Viewer

Several flow cytometry companies, including BD Biosciences and ThermoFisher Scientific, have created fluorochrome spectral viewers to facilitate panel design and have a theoretical prediction of the compensation. These online tools are designed to aid customer understanding of important principles, dependencies, and relationships involved in selecting fluorochromes for multicolor flow cytometry. Users of these tools should ultimately rely on their own judgment when selecting fluorochromes for multicolor panels and experiments.

Panel Designer

One difficult task that people encounter when they start making multicolor flow cytometry panels is the process of finding antibodies and determining which fluorochromes to use for our instruments. The CRI Flow Cytometry Facility recommends using Fluorish 2.0 or Fluorofinder to help you in this task.

Setting up a Flourish account:

  1. Go to the Flourish website using this specific link.
  2. Enter your information and in the institution box, make sure the following appears: “Children’s Research Institute at UT Southwestern.” When done, click the “Register” button.

Setting up a Fluorofinder account:

  1. Go to http://cri-utsw.fluorofinder.com
  2. “Create an account” if you want your panel to be saved.
  3. Select the instrument you want to use, and follow the instructions.

Cytobank

Cytobank provides a turnkey storage solution for your flow cytometry data. Cytobank allows researchers a new way to manage, search and analyze their FCS files over the web. The analysis system is designed to incorporate annotations and make it easier to create figures for lab meetings, publications or sharing. Cytobank also facilitates discussions about flow cytometry across and within groups. Users can share experiment data and details quickly and easily through the web. Collaborators can start with the result and drill down to the underlying single cell data.

A key challenge in flow cytometry is communicating the results along with sufficient experiment details to allow for additional analysis. In Cytobank, users provide these details during the course of analysis, allowing you to start with analyzed or gated data, drill down to the raw data and export gated or raw data in tab-delimited or fcs formats.

Contact

Nicolas Loof, MS

Nicolas Loof, MS

Head of the Moody Foundation Flow Cytometry Facility


Carolyn Diaz, BS

Carolyn Diaz, BS

Flow Cytometry Operator