Flow Cytometry

flow cytometry equipmentThe Flow Cytometry Core's primary function is to offer Center Investigators the ability to utilize complex and technically-challenging flow cytometric approaches to characterize cells and their functional and signaling properties.

The Core is also committed to an educational goal. Not only will Center members be trained on the operation of instruments, it is the intent that all users completely understand cytometric theory, application, and data analysis.

 


Aims of the Core

  1. Support current COBRE and Center projects. The Core provides state-of-the-art equipment and the technical and analytical expertise to address the flow cytometric needs of the supported early career investigators, other members of the Diabetes and Obesity Center and other investigators at the University of Louisville.
  2. Educate and train investigators. Many of our Cores organize seminars and informal meetings to serve as a forum in bringing together users and to initiate discussion of novel reagents and applications. It is intended that all COBRE-supported early career investigators, in their transition to independence, will completely understand cytometric theory, application, and data analysis and be able to competently incorporate flow cytometry as an analytical tool in their future studies.
  3. Develop new protocols and techniques. The Core is currently expanding its operations to support the multidimensional flow cytometry needs in the future. With the incorporation of new investigators into the Center in the future,new projects and experiments will be carried out to perform cutting-edge research to address the rapidly spreading epidemics of diabetes and obesity.

Facilities & Instrumentation

MoFlo cell sorterMain facilities of The Core are housed in a 600 ft2laboratory in the Cadiovascular Innovation Institute (CII) building (302 East Muhammad Ali Blvd., CII, Room 214, Louisville, KY 40202) at the Health Science Center (HSC) at the University of Louisville.

This lab is fully equipped with LSR II and MACSQuant® cytometers (described below) centrifuges, computers, refrigerators and freezers, tissue culture hood for sterile techniques, incubators, and all the small equipment needed for cell handling and staining.

In addition, the Core maintains a Fortessa in the Baxter II building on the HSC campus.

Details of these instruments are as follows:

  • LSRII - This Becton Dickinson flow  cytometer is kept in room 214 of the CII. It is equipped with four(4) solid-state lasers; blue (488 nm) , red (633 nm), violet (405 nm), and ultraviolet (325nm). It has the capability to detect up to 18 colors six off the 488 and 405 lasers, 4 off the 633 laser and 2 off the UV laser.
  • MACSQuant® - This Miltenyi instrument is also housed in Room 214 of the CII building. It has a 488nm laser (4-color detection), 638nm laser (2-color detection), and a 405nm laser (2-color detection). In addition, it is equipped for 24 tube and 96 well plate automation.
  • Fortessa - This BD instrument is housed in the Baxter II building. It is equipped with 4 lasers: a 488nm (2-color detection), a 640nm (3-color detection), a 405nm (6-color detection), and a 561nm (4-color detection). It also has high throughput capability. 

In addition to the flow cytometers, the Core maintains other equipment for general use. These include:

  • Miltenyi gentleMACS™ Dissociator - This instrument performs standard tissue digestions for the generation of uniform single cell preparations.
  • MAGPIX® - This instrument uses Luminex's xMAP® technology for multiplex analysis of plasma samples.

Our Expertise

Jason Hellmann, Ph.D.

  • multi-color panel design
  • tissue digestion and single-cell isolation techniques
  • identification, quantification, and functional assessment of circulating and tissue resident immune cells

Timothy O'Toole, Ph.D.

  • identification of rare cell types and microparticles
  • design and use of multi-color panels
  • analysis of platelet function

Marcin Wysoczinski, Ph.D.

  • quantification of neutrophils, monocytes/macrophages, B and T cell, and their discrete subsets in peripheral blood, spleen, bone marrow, and heart tissue
  • quantification of bone marrow hematopoietic stem and progenitor cell sub-populations

Marina Malovichko, Ph.D.

  • multi-color panel design
  • analysis of pulmonary immune cells