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What is Phosphoflow?

Cells are continuously responding to their microenvironment through the modulation of cellular signaling pathways that control a multitude of responses. Phosphoflow is a specialized application of flow cytometry that enables researchers to examine the phosphorylation status of specific proteins that govern cellular responses through signaling pathways (summarized in Fig. 1). It has been demonstrated that dysfunction of these cell signaling pathways can play a significant role in the advancement of disease across many areas of medicine, from the disruption of immune responses to microbial infection, to the development of autoimmune diseases and cancer. As such, the underlying mechanisms of phosphorylation and dephosphorylation of these regulatory signaling proteins are now being targeted for therapeutic intervention.

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Figure 1. Principles of Phosphoflow Staining Technique for Flow Cytometry. (1) Heterogeneous samples of cells are treated with two different stimuli, A and B, or a combination of the two A+B -this stimulation activity has the potential to induce phosphorylation of critical regulatory target proteins within the cells. (2) After stimulating, the cells are then fixed, permeabilized, and stained with antibodies that bind only to the phosphorylated forms of these target proteins. (3) Cells are analyzed on a flow cytometer, enabling the detection and measurement of phosphorylated forms of these regulatory target proteins within different cell types.

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Cell Signaling Mechanisms

At its core, cell signaling is mediated through the modulation of specific proteins. Phosphorylation is the addition of a phosphate group to the polar group on various amino acids- principally, serine, threonine, and tyrosine through the activity of highly specific kinase enzymes. The opposite reaction, dephosphorylation, is the removal of a phosphate group via a phosphatase enzyme.

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Within the human proteosome, there are over
200,000 known phosphorylation sites
and up to 30% of proteins may be modified through kinase and phosphatase activity.

These include a diverse array of functional proteins from membrane-bound receptors, ion channels, and transporters, to cytosolic enzymes, cell signaling, protein degradation associated proteins, and nuclear transcription factors and histones.

When it comes to interrogating cell signaling mechanisms, phosphoflow provides unparalleled granularity, enabling researchers to decipher the responding cell populations and monitor these mechanisms in detail.

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Considerations For Successful Phosphoflow

Cell signaling functions to transduce signals from the extracellular environment, to generate a response within the intracellular environment. Cell signaling pathways can be very complex and create intermittent cascade reactions that result in several phenotypic changes throughout the cell. These are both highly specific and transient in nature, and therefore there are several important considerations when designing any phosphoflow experiment. Working with the experienced team at FlowMetric will ensure the optimal experimental approach for your phosphoflow program goals.

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Timing is everything.

There are a series of proteins that are transiently phosphorylated within a signaling pathway, and so understanding the timing of these events is critical if they are to be detected with phosphoflow before either dephosphorylation or protein degradation is triggered.

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Selection of the target protein epitope.

Many of the target proteins have multiple potential phosphorylation sites, that may be regulated very differently. Make sure that your antibody conjugate is targeting the correct epitope for the signal-specific phosphorylation. Always ensure that any conjugate used for any flow cytometry application have been validated for this use.

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Sample processing considerations.

Phosphoflow requires the fixation and permeabilization of the cell. Permeabilization is a balance of gaining access to the intracellular compartment, versus the loss of protein targets. Fixation conditions can impact the landscape of the cell for phosphoflow detection. All of these parameters should be evaluated for their effect on activation specific phospho-signal.

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Optimizing the order of fixation, permeabilization and staining.

In many cases, phosphoflow is performed in conjunction with CD marker staining to identify specific cell populations. It is important to carefully access the binding of conjugates to their target epitopes under different processing conditions- CD cell lineage staining may be performed prior to fixation, or after permeabilization.

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Staining controls are crucial.

Unstimulated negative and stimulated positive controls, as well as compensation controls (or BD CompBead Particles) are all recommended. As will all flow analysis, a viability dye should be used: fixable viability dyes such as Ghost dyes are a great option for phosphoflow.

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Pre-stimulation.

Baselines for phosphoflow analysis can be tricky, so it is important to evaluate and be consistent with pre-stimulation conditions -although cells shouldn’t be starved, changing media the day of stimulation can alter signaling, and cells should be rested for ~90 minutes at 37˚C before stimulation.

Critical Experimental Design

There are various commercial options for phosphoflow buffers and related protocols. The FlowMetric quality driven approach ensures that we follow good practice techniques including using a fresh lot of permeabilization and fixation buffers for each experiment and ensuring that conjugate binding- either to phospho-epitopes or CD cell lineage markers are not impacted by these buffers. Fluorophores are selected for their optimal performance in intracellular staining, and compatibility with fixation.

Typically, the first step of any phosphoflow analysis following cell signal activation is the fixation of the cells either using formaldehyde or methanol. At FlowMetric this is assessed for each phospho-epitope target since there are distinct nuances in how epitopes are presented for labeling after fixation. For example, Stat1 is best assessed under methanol fixation.

The selected permeabilization process is evaluated for the cell populations of interest, as well as the location of the target protein (cytosolic, nuclear, organelle). Saponin and digitonin are used to permeabilize the plasma membrane for the labeling of cytosolic target proteins, however their use is not conducive with labeling organelle- or nuclear-localized proteins. In these instances, Triton X-100 or Tween 20 are recommended, and all permeabilization procedures are carefully assessed before use.

Phosphoflow promises enormous potential for studying the immune system and the complex responses in disease states. But it can be a challenging experimental system to optimize, and as such, it is important to empirically test and validate your system of buffers and conjugates to ensure the highest quality of phosphoflow analysis.

Applications of Phosphoflow

Drug Discovery

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Profile cell signaling responses to lead compounds

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Direct analysis of up- and down-regulation of pathways in animal models

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Establish cell signaling profiles for therapeutic targeting

High-Throughput Screening

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Multiplexed signaling pathway analysis

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In vitro drug safety and efficacy screening

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Fluorescent Cellular Barcoding

Preclinical and Clinical Trials

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In vivo drug efficacy and safety screening

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Cellular assays to monitor clinical endpoints

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Support PK/PD

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Mechanism of Action

Diagnostics and Therapeutic Monitoring

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Identify disease-specific cellular responses

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Application of precision medicine approaches to disease management

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Implement prognostic markers


Case Study

In the example below, we examined key phosphoproteins within lymphocyte subsets without prior isolation from whole blood.

Fresh whole blood from normal healthy donors was stimulated with either hIL-6 or hIL-2 at a concentration of 100ng/mL for 15 minutes at 37°C. The phosphoflow process to measure STAT-3 and STAT-5 included the lysis and fixation, washing, and subsequent permeabilization of the cells for 30 minutes at 4°C. The cells were then stained for the surface markers CD3, CD4, CD8, and CD19 for one hour at room temperature, before acquisition on a BD Fortessa™.

The plots show the CD4+ and CD8+ T cell signaling profiles for each of the phosphorylation markers, STAT-3 and STAT-5. As expected, the CD19+ B-cells were negative for these markers.

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Figure 2. The first plot shows a light scatter gate used to identify peripheral blood cell populations. Next, a CD3+ and CD19+ population is identified. Finally, CD8+ and CD4+ T Cell populations are identified within the CD3+ population.

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Figure 3. Activation profiles for CD4+ and CD8+ T cells as well as CD19+ B cells. The data are displayed as histogram overlays with untreated cells. These histograms show signaling responses of each phosphorylation marker, STAT-3 and STAT-5, induced by the corresponding response modifier, hIL-6 (100ng/mL) for STAT-3 and hIL-2 (100ng/mL) for STAT-5.

The FlowMetric Advantage

Phosphorylation is a complex cell signaling event, but measuring phosphorylation doesn’t have to be. FlowMetric has sourced key reagents and designed and optimized a variety of multiplexed phosphoflow panels over the past 12 years. Many of these panels have been implemented to analyze clinical samples, requiring careful development and critical evaluation to ensure robust assay performance. The FlowMetric team has leveraged their experience to interrogate cell signaling pathways in many model systems and cell types. Armed with this information, we can help ensure that confident decisions can be made for the progression of your biomedicals through clinical development.

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