Flow cytometry has come of age; it is now being used as a tool for systems biology research based on its unique ability to monitor a large number of parameters within individual cells.
At the recent ELRIG conference “BioPharmaceuticals Flow Cytometry & Imaging”, speakers presented their solutions to the challenges faced by researchers that are looking to exploit the resolution of flow cytometry to address biological questions within the context of systems biology complexity.
Driven by the desire to use the flow cytometer as a high-throughput, high-content instrument for the screening of cell-signaling pathways in single cells, Bernd Bodenmiller, Ph.D., group leader at the University of Zurich, and his lab employ mass cytometry, a novel technology that increases the number of parameters that could be resolved from 10–12 to up to 100.
During his postdoctoral time at Stanford University, Dr. Bodenmiller and colleagues developed a technology called mass-tag cellular barcoding (MCB), which enabled them to pool cell subpopulations plated in a 96-well plate.
Each plate position contained a unique set of stable metal isotopes that can be used to label cells in each well. In a process not unlike the labeling of cells with fluorescently tagged antibodies, the mass tags are introduced into the cells via a small molecule compound, which binds the metal isotopes and covalently binds to cellular proteins.
These metal tags (barcodes) can then be used in the mass cytometer to identify the source of the cell under interrogation using fluorescent tags. In the mass cytometer, only seven channels are needed to resolve the barcodes from a 96-well plate, which leaves more than 30 channels to resolve cell-surface markers and differentiate cellular markers from intracellular epitopes.
“The example we use to illustrate the power of this approach is the interrogation of phosphorylation patterns in peripheral blood monocytes,” explained Dr. Bodenmiller.
“We are able to differentiate phosphorylation patterns in 14 different cell types, looking at 14 different phosphorylation sites after exposing the cells to small molecule kinase inhibitors that target the active site in the kinase (e.g., rapamycin) provided in eight different drug concentrations and 12 different cellular conditions, which yields 18,816 measured phosphorylation levels in a single measurement.”
What the lab has been able to learn from this exercise is that some expected patterns of phosphorylation can be observed. Specifically, closely related cell types respond similarly to inhibition by known compounds based on previous analysis using these cell types and the particular drugs under analysis. But in addition, novel patterns were revealed, which allows the lab to group unknown compounds with known ones with respect to mechanism of inhibition and the site of action.
“The other benefit of using MCB prior to antibody staining with fluorescent tags is that throughput goes up to thousands of samples per day, the costs of antibody are significantly reduced, and the quality of the data increases due to homogenous cell labeling,” concluded Dr. Bodenmiller.