We are working on a number of important aspects of antigen presenting cell biology in relation to processing and presenting glycan antigens by MHC class II proteins. As such, we are conducting cutting-edge confocal microscopy studies designed with live and fixed cell imaging. The lab owns a Leica SP5 broadband confocal microscope with the standard and resonant scan heads (regular and high speed imaging). Below is a picture of our system:
We also use a Leica Laser Capture Microdissection instrument (housed in the same room as our confocal) for isolation of cells and molecules from defined regions of tissues for Western blotting and quantitative rtPCR.
A number of technologies are available for the study of a variety of biochemical and cellular processes. In vitro binding and ELISA-based studies are carried out using time-resolved fluorescence on the Wallac Victor3V (from Perkin Elmer) spectrometer. Our system (with a plate washer) is shown below:
This instrument will also read absorption (UV/Vis), standard fluorescence intensity, and luminescence. Samples are analyzed in microplate format (from 6 well to 1024 well formats), enabling high throughput analysis if needed.
The Victor3V will also enable analysis of cellular activity in live cells using fluorescence resonance energy transfer as an end point. This instrument is easy to use, computer controlled, and a center piece of many biochemical and cellular assays we perform.
Fluorescence-Activated Cell Sorting (FACS)
The Cobb lab has an Accuri C6 Flow Cytometer for our own routine use, independent of the core facilities. This system is a dual-laser, four-color instrument that will greatly facilitate the lab's throughput. We will use this instrument in most of the projects currently underway. We also make use of the Case Comprehensive Cancer Center Flow Core, in which we utilize the FACSAria for sterile cell sorting.
Accuri C6 Flow Cytometer
High-Pressure Liquid Chromatography (HPLC)
We use chromatography for purification and analytical purposes. For all of these needs, we use a GE Healthcare Akta Purifier 10 HPLC. For carbohydrate separations, the system is fitted with a refractive index monitor for the detection of molecules that do not absorb light like most proteins. We have two fraction collectors, with one in the chromatography refrigerator and one on the bench. Likewise, some of the columns are mounted in the refrigerator. The main part of our system is shown below:
We have been working with murine asthma models for a number of years. A recent addition to our arsenal of tools is a FlexiVent lung function instrument from SciReq. This instrument allows for detailed analyses of lung function, including resistance and compliance. We can also use the instrument for methacholine challenges to quantify hyperresponsiveness in mice. See the SciReq site for much more information of this instrument's capabilities.