Using Phospho-Flow Cytometry to Study Signaling in Human PBMCs
Why Study Signaling at the Single-Cell Level
Every immunologist agrees that immune cells are heterogeneous. Human peripheral blood mononuclear cells (PBMC) contain many different immune cell types – CD4+ T cells alone exist in dozens of uniquely functioning subsets.
Cells communicate through receptor-mediated signaling in response to cytokines, chemokines, various receptor ligands, and engagement of the T cell or B cell receptors. These responses are what differentiate the many immune cell types found in human PBMC. But averaging across a complex PBMC population obscures the relationship between protein expression and signaling states in individual cell populations, and at the single-cell level.
What Is Phospho-Flow Cytometry?
Dr. Gary Nolan and colleagues pioneered a method that uses flow cytometry to examine cell signaling in single cells, using antibodies that target phosphorylated protein sites. This method is called phospho-flow cytometry, or “phosflow.”
In phosflow, PBMCs or other cell populations are stimulated with signaling receptor ligands and/or antagonists for a set period of time. The cells are then fixed using paraformaldehyde-based buffers, which locks them into their induced phosphorylation state. Next, the cells are permeabilized and stained with fluorescently-labeled antibodies against the phosphorylated proteins, cell-identifying antigens, and other proteins of interest, then analyzed on a flow cytometer.
Using this method, our lab has studied the response to many cytokines by analyzing phosphorylation of the STAT family of transcription factors. We’ve also studied phosphorylation events downstream of T cell receptor (TCR) and B cell receptor (BCR) engagement, and Toll-like receptor ligation, including NF-kB (p65), ERK, p38, and ZAP70. BD Biosciences is a key source for many of the reagents these assays need, offering a range of buffers and antibodies against important signaling targets.
Optimizing the Protocol: Permeabilization
There are several considerations for optimizing this protocol. The first is choosing the right permeabilization method. Nolan and colleagues published a protocol using 100% methanol for permeabilization after fixation, and this is the method used most often.
One key advantage of this method: after methanol permeabilization, cells can be stored for an extended period at -20°C or -80°C before antibody staining and flow cytometry. This means many samples can be stimulated on different days, while the flow cytometry analysis is run later in one large batch, which helps limit variability in staining intensity between experiments.
This matters because in phosflow data, the fluorescence intensity – which reflects the magnitude of protein phosphorylation – is often the main thing being analyzed, though the percentage of cells that respond to a given signal can also be an important measure. A number of commercially available buffer sets exist for these assays; choose one based on the phospho-proteins and other antigens you’re targeting.
Optimizing the Protocol: Antigen Selection
Another important factor is choosing the right target antigens to identify cell populations of interest. Some antigens do not survive permeabilization with certain reagents, including methanol. For example, CD19 (for identifying B cells) and CD14 (for monocytes) have not worked well in our hands. CD20 and CD33 are effective alternatives that hold up well with methanol.
Cytobank, an online resource for phosflow created by Gary Nolan’s lab, is a helpful tool for selecting the right antigens and antibody clones. It lists antibodies, including phospho-target antibodies, that have been tested (successfully and unsuccessfully) with BD Biosciences’ various permeabilization buffers.
Summary
Phosflow is an excellent method for analyzing signal transduction in complex cell populations. Single-cell assays for protein expression and signaling will keep advancing our understanding of the immune system’s complexity.
Nolan lab Phosflow protocol:
Single-cell phospho-protein analysis by flow cytometry. Schulz KR, Danna EA, Krutzik PO, Nolan GP.Curr Protoc Immunol. 2012 Feb;Chapter 8:Unit 8.17.1-20.
BD Biosciences Phosflow Homepage:
https://www.bdbiosciences.com/research/phosflow/index.jsp?WT.ac=FP_Phosflow
Cytobank’s list of antibodies tested with various permeabilization buffers:
https://www.cytobank.org/facselect/
Further Reading:
Flow cytometric analysis of cell signaling proteins. Suni MA, Maino VC. Methods Mol Biol. 2011;717:155-69.
Single-cell, phosphoepitope-specific analysis demonstrates cell type- and pathway-specific dysregulation of Jak/STAT and MAPK signaling associated with in vivo human immunodeficiency virus type 1 infection. Lee AW, Sharp ER, O’Mahony A, Rosenberg MG, Israelski DM, Nolan GP, Nixon DF. J Virol. 2008 Apr;82(7):3702-12.
