Customer Education

Multispectral Imaging Flow Cytometry: Illuminating T cell APC Interactions

 What Does it Cover?

Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging by fluorescence microscopy. However, immunological synapses are rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis brand imaging flow cytometers to objectively collect imagery of large numbers of cells.  We report the percentage of T cells involved in an organized immunological synapse, the recruitment of adhesion molecule LFA-1 and signaling molecule Lck to the synaptic complex and subsequent translocation of NFkB from the cytoplasm to the nucleus in the T cell. In this study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB) were incubated with human T cells to create T cell-APC conjugates. Cells were stained in various combinations for CD3, CD19, Actin, LFA-1, Lck and NFkB.  Results from the FlowSight and the ImageStream imaging flow cytometers are compared. Using the FlowSight imaging flow cytometer we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized immunological synapse in an objective and statistically significant manner. Employing the ImageStream imaging flow cytometer we further evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex in T cells and measure the nuclear localization of NFkB in the T cell.


 What Will You Learn?

  • Amnis brand imaging flow cytometers principles of operation
  • Isolating conjugates for immunological synapse analysis
  • Identifying/masking the immunological synapse in T cell-APC conjugates
  • Evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex
  • Measure the nuclear localization of NFkB in the T cell-APC conjugates

 Who Should Attend?

  • Immunologists
  • Biologists
  • Cancer biologists
Speaker Bio
Haley R. Pugsley, Ph.D
Senior Research Scientist
Haley received her Ph.D. in Analytical Chemistry from the University of Washington working under the direction of Professor Norman J. Dovichi. At the University of Washington she used capillary electrophoresis to study protein fingerprinting of breast cancer tissue and created two reporter cell lines to investigate the expression of RecA in Deinococcus radiodurans. Dr. Pugsley did her postdoctoral fellowship at Hematologics, Inc. under the direction of Dr. Michael R. Loken where she focused on clinical flow cytometry and its use in diagnosing hematologic malignancies. Dr. Pugsley joined Amnis in 2011 developing applications for both the ImageStreamX Mark II and FlowSight imaging flow cytometers. Dr. Pugsley has worked on many applications including autophagy, immunological synapse, algae biomass, cell signaling, exosomes and most recently an automated method for leukemia detection using multi-parametric analysis of imaging flow cytometry data.