This work was conducted in the lab of Professor Kjetil Task&#233;n, and was supported by the Norwegian Cancer Society and Stiftelsen Kristian Gerhard Jebsen. Johannes Landskron and Marianne Enger are acknowledged for critical reading of the manuscript.

Blood samples were received following written informed consent from all donors. The study was approved by the Regional Committee for Medical and Health Research Ethics of South-East Norway and the research on human blood was carried out in accordance with the Declaration of Helsinki8.
NOTE: Steps 1-3 should be performed under sterile conditions in a tissue culture hood.
1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from CLL Patient Blood Samples
CAUTION: Human blood should be handled according to regulations for Biosafety Level 2.
<ol>
	<li>Dilute the blood 1:1 with phosphate-buffered saline (PBS: 136.9 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4 x 2H2O, 1.8 mM KH2PO4, pH 7.4) and transfer to 50 mL tubes (30 mL/tube).</li>
	<li>Carefully layer 10 mL of a density gradient medium (e.g., Lymphoprep) to the bottom of the tube using a 10 mL pipette.</li>
	<li>Centrifuge at 800 x g for 20 min at 4 &#176;C. The PBMCs are now visible on top of the density gradient medium layer.</li>
	<li>Use a Pasteur pipette to transfer the cells into two new 50 mL tubes. Wash twice with PBS (fill up the tubes).</li>
	<li>Centrifuge at 350 x g for 15 min. Discard the supernatant and resuspend in 3 mL of PBS.</li>
	<li>Count the cells using a preferred method.</li>
	<li>Centrifuge the cells at 350 x g for 5 min. Discard the supernatant.&#160;NOTE:&#160;Steps 1.8 to 3.2 are optional. It is possible to proceed directly to step 3.3.</li>
	<li>Resuspend the cells in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) and freeze down in suitable aliquots using cryo tubes. 
	NOTE: DMSO is toxic to the cells. Work fast once the cells are mixed with FBS/DMSO. Cells can be stored long term in liquid nitrogen.</li>
</ol>
2. Thawing of Cells
<ol>
	<li>Quickly thaw the cells in a 37 &#176;C water bath. 
	NOTE: DMSO is toxic to the cells. Work fast to limit the exposure to DMSO.</li>
	<li>Wash the cells once with 10 mL of cold Roswell Park Memorial Institute medium (RPMI 1640 with supplemental GlutaMAX, see Table of Materials).</li>
	<li>Centrifuge at 300 x g for 5 min. Discard the supernatant.</li>
	<li>Resuspend the cells in RPMI 1640 medium supplemented with sodium pyruvate, MEM non-essential amino acids and penicillin/streptomycin (added at 1x dilution according to instructions) and 10% FBS. Transfer the cells to a small cell culture flask and leave in an incubator at 5% CO2, 37 &#176;C for 1 hour to allow the cells to calibrate.</li>
</ol>
3. Preparation of Cells
<ol>
	<li>Count the viable cells using a preferred method.</li>
	<li>Transfer the cells to a 50 mL tube and centrifuge at 300 x g for 5 min. Discard the supernatant.</li>
	<li>Resuspend the cells in RPMI 1640 medium (step 2.2) supplemented with 1% FBS to no more than 50 x 106 cells/mL.</li>
	<li>Transfer the required amount of cell suspension to wells in a 96 well V-bottom plate. 
	NOTE: Number of wells corresponds to the number of conditions to be tested. Samples for a stimulation time-course are drawn from a single well. Calculate 50 &#181;L sample per time-point + 50 &#181;L of dead volume. Save samples for compensation controls (one unstained sample + one sample per barcoding dye).</li>
	<li>Transfer the 96 well plate to a pre-heated 37 &#176;C water bath. Rest the cells for 10 min.</li>
</ol>
4. Stimulation and Fixation of Cells
NOTE: Perform steps 4-8 on the lab bench (i.e., not sterile).
CAUTION: The main ingredient of Fix Buffer I is paraformaldehyde, which is toxic (inhalation and skin contact). Handle with care.
<ol>
	<li>Prepare a 96 well V-bottom plate with 60 &#181;L of Fix Buffer I per well per sample. Leave in the 37 &#176;C water bath. 
	NOTE: Cells: Fix buffer should be 1:1. In order to allow for evaporation at 37 &#176;C, the Fix buffer is initially in abundance.</li>
	<li>Optionally, treat the cells with drugs before stimulation.</li>
	<li>Transfer a 50 &#181;L control sample to the fix plate. Mix by pipetting up and down.</li>
	<li>Optionally, start the stimulation time-course by adding 10 &#181;g/mL anti-IgM to the cells. Mix by pipetting up and down.</li>
	<li>Transfer a 50 &#181;L sample to the fix plate at each time-point. Mix by pipetting up and down. 
	NOTE: Anti-IgM induced signaling is usually initiated early (minutes).</li>
	<li>Leave the fix plate at 37 &#176;C for 10 min after the last sample has been added.</li>
</ol>
5. Fluorescent Cell Barcoding (FCB)
NOTE: See Table 1 for a list of barcoding reagents.
<ol>
	<li>Wash the fixed cells 3x with PBS (fill up the wells).</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant.</li>
	<li>Prepare a 96 well V-bottom plate with barcoding reagents. Pipet 5 &#181;L of each barcoding reagent per well in the number of combinations required to stain all samples following the staining matrix, e.g., in Figure 1B. Each sample will have a unique combination of different barcoding concentrations.</li>
	<li>Resuspend the cells in 190 &#181;L of PBS and transfer to the barcoding plate. Mix thoroughly. 
	NOTE: Stain one compensation sample with the highest final concentration used for each barcoding reagent and save one unstained sample.</li>
	<li>Leave the cells for 20 min at room temperature, in the dark.</li>
	<li>Wash the stained cells 2x with flow wash (PBS, 1% FBS, 0.09% sodium azide) (fill up the wells).</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant.</li>
	<li>Add 190 &#181;L of flow wash to the cells and combine the barcoded samples in one 15 mL tube. Transfer each compensation control to a separate 1.7 mL tube.</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant.</li>
</ol>
6. Cell Permeabilization for Intracellular Antigen Staining
CAUTION: The main ingredient of Perm Buffer III is methanol which is toxic (inhalation and skin contact) and flammable. Handle with care.
<ol>
	<li>Transfer 2 mL of Perm Buffer III to a 15 mL tube. Leave at -20 &#176;C so it is ice-cold upon use. 
	NOTE: The Perm Buffer can be left at -20 &#176;C from the start of the experiment.</li>
	<li>Add 1.5 mL of ice-cold Perm Buffer to the barcoded cell population (in a 15 mL tube) and 100 &#181;L to each compensation control (in 1.7 mL tubes) drop-wise while vortexing to avoid that the cells clump together.</li>
	<li>Transfer the cells directly to -80 &#176;C. Leave for a minimum of 30 min. 
	NOTE: It is natural to pause the experiment at this point. Cells in Perm Buffer can be stored long term at -80 &#176;C.</li>
</ol>
7. Antibody Staining
NOTE: See Table of Materials for a list of reported phospho-specific antibodies.
<ol>
	<li>Transfer the cells from -80 &#176;C to a box of ice.</li>
	<li>Wash 3x with flow wash. 
	NOTE: It is important to add flow wash in excess to see the cell pellet, e.g., add 3 mL of flow wash to the barcoded cell population and 1 mL to each compensation control.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the barcoded cell population in a volume of flow wash, which allows 25 &#181;L of cell suspension per phospho-antibody stain. Resuspend the compensation controls in 200 &#181;L of flow wash.</li>
	<li>Prepare antibodies for staining in a 96 well V-bottom plate. The final volume will be 50 &#181;L/well. Per well, add phospho-specific antibody diluted in flow wash to a final volume of 10 &#181;L, surface marker diluted in flow wash to a final volume of 15 &#181;L, and 25 &#181;L of cell suspension. 
	NOTE: Antibody dilutions should be titrated prior to the experiment. Include isotype control.</li>
	<li>Leave the cells for 30 min at room temperature, in the dark.</li>
	<li>Wash the stained cells 2x with flow wash (fill up the wells).</li>
	<li>Centrifuge at 500 x g for 5 min. Discard the supernatant.</li>
	<li>Resuspend the cells in 150 &#181;L of flow wash.</li>
</ol>
8. Preparation of Compensation Controls
<ol>
	<li>Prepare compensation controls for the antibody-conjugated fluorochromes in parallel with the antibody staining. Use compensation beads according to the vendor&#8217;s instructions.</li>
</ol>
9. Flow Cytometry Analysis
NOTE: The experiment can be run on a flow cytometer with a High Throughput Sampler (HTS).
<ol>
	<li>Optimize the photomultiplier tube (PMT) voltage with the unstained control.</li>
	<li>Run compensation controls and calculate the compensation matrix.</li>
	<li>Run samples. The event rate should be in accordance with the instrument specifications.</li>
</ol>
10. Gating Strategy and Data Analysis
<ol>
	<li>Import the FCS files from the experiment to a flow cytometry analysis software like FlowJo or Cytobank (https://cellmass.cytobank.org).</li>
	<li>Gating strategy
	<ol>
		<li>Select lymphocytes by plotting SSC-A versus FSC-A in a density dot plot.</li>
		<li>Display the lymphocytes and select the singlets by plotting SSC-A versus FSC -W.</li>
		<li>Display the single cells and gate the cell type by plotting SSC-A versus the surface marker.</li>
		<li>Display the cell type population in a Pacific Blue versus SSC-A density plot and select the different FCB populations based on their Pacific Blue staining intensity (see Figure 1A).</li>
		<li>Plot the phospho antibody channel against the FCB channel, or as a heatmap (see Figure 1A) to display the phosphorylation events.</li>
	</ol>
	</li>
	<li>Calculate phospho-signals using the inverse hyperbolic sine (arcsinh) of the MFI (median fluorescent intensity) of phospho-signal versus isotype control (basal phosphorylation levels, see Figure 1D), or of stimulated versus unstimulated cell populations (see Figure 1E).</li>
</ol>

<ol>
	<li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+reverse-phase+protein+arrays+as+pharmacodynamic+assays+for+functional+proteomics,+biomarker+discovery,+and+drug+development+in+cancer.">Using reverse-phase protein arrays as pharmacodynamic assays for functional proteomics, biomarker discovery, and drug development in cancer.</a> Seminars in Oncology. 43 (4), 476-483 (2016).</li><li>Krutzik, P. O., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cell+barcoding+in+flow+cytometry+allows+high-throughput+drug+screening+and+signaling+profiling.">Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.</a> Nature Methods. 3 (5), 361-368 (2006).</li><li>Myhrvold, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+profiling+of+phospho-protein+levels+in+chronic+lymphocytic+leukemia.">Single cell profiling of phospho-protein levels in chronic lymphocytic leukemia.</a> Oncotarget. 9 (10), 9273-9284 (2018).</li><li>Parente-Ribes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+tyrosine+kinase+inhibitors+reduce+CD40L-induced+proliferation+of+chronic+lymphocytic+leukemia+cells+but+not+normal+B+cells.">Spleen tyrosine kinase inhibitors reduce CD40L-induced proliferation of chronic lymphocytic leukemia cells but not normal B cells.</a> Haematologica. 101 (2), e59-e62 (2016).</li><li>Blix, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospho-specific+flow+cytometry+identifies+aberrant+signaling+in+indolent+B-cell+lymphoma.">Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma.</a> BMC Cancer. 12, 478 (2012).</li><li>Irish, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+profiling+of+potentiated+phospho-protein+networks+in+cancer+cells.">Single cell profiling of potentiated phospho-protein networks in cancer cells.</a> Cell. 118 (2), 217-228 (2004).</li><li>Myklebust, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+B-cell+receptor+signaling+in+non-Hodgkin+lymphomas+identified+by+single-cell+profiling.">Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling.</a> Blood. 129 (6), 759-770 (2017).</li><li>World Medical Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Medical+Association+Declaration+of+Helsinki:+ethical+principles+for+medical+research+involving+human+subjects.">World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects.</a> THE JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 310 (20), 2191-2194 (2013).</li><li>Siveen, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+STAT3+signaling+pathway+in+cancer:+role+of+synthetic+and+natural+inhibitors.">Targeting the STAT3 signaling pathway in cancer: role of synthetic and natural inhibitors.</a> Biochimica et Biophysica Acta. 1845 (2), 136-154 (2014).</li><li>Fabbri, G., Dalla-Favera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+pathogenesis+of+chronic+lymphocytic+leukaemia.">The molecular pathogenesis of chronic lymphocytic leukaemia.</a> Nature Reviews Cancer. 16 (3), 145-162 (2016).</li><li>Arnason, J. E., Brown, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+B+Cell+Signaling+in+Chronic+Lymphocytic+Leukemia.">Targeting B Cell Signaling in Chronic Lymphocytic Leukemia.</a> Current Oncology Reports. 19 (9), 61 (2017).</li><li>Landskron, J., Tasken, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoprotein+Detection+by+High-Throughput+Flow+Cytometry.">Phosphoprotein Detection by High-Throughput Flow Cytometry.</a> Methods in Molecular Biology. 1355, 275-290 (2016).</li><li>Krutzik, P. O., Clutter, M. R., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+analysis+of+murine+immune+cell+surface+markers+and+intracellular+phosphoproteins+by+flow+cytometry.">Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.</a> Journal of Immunology. 175 (4), 2357-2365 (2005).</li><li>Pollheimer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-33+drives+a+proinflammatory+endothelial+activation+that+selectively+targets+nonquiescent+cells.">Interleukin-33 drives a proinflammatory endothelial activation that selectively targets nonquiescent cells.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (2), e47-e55 (2013).</li><li>Erts&#229;s, H. C., Nolan, G. P., LaBarge, M. A., Lorens, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsphere+cytometry+to+interrogate+microenvironment-dependent+cell+signaling.">Microsphere cytometry to interrogate microenvironment-dependent cell signaling.</a> Integrative biology: quantitative biosciences from nano to macro. 9 (2), 123-134 (2017).</li><li>Lin, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+phospho-specific+flow+cytometry+can+detect+dynamic+changes+of+phospho-Stat1+level+in+lung+cancer+cells.">Single cell phospho-specific flow cytometry can detect dynamic changes of phospho-Stat1 level in lung cancer cells.</a> Cytometry A. 77 (11), 1008-1019 (2010).</li><li>Simmons, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry-based+single-cell+analysis+of+intact+epithelial+signaling+reveals+MAPK+activation+divergent+from+TNF-alpha-induced+apoptosis+in+vivo.">Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-alpha-induced apoptosis in vivo.</a> Molecular Systems Biology. 11 (10), 835 (2015).</li><li>Simmons, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+coordination+between+signaling+pathways+is+revealed+in+human+colorectal+cancer+using+single-cell+mass+cytometry+of+archival+tissue+blocks.">Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks.</a> Science Signaling. 9 (449), rs11 (2016).</li><li>Friedman, A. A., Letai, A., Fisher, D. E., Flaherty, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+medicine+for+cancer+with+next-generation+functional+diagnostics.">Precision medicine for cancer with next-generation functional diagnostics.</a> Nature Reviews Cancer. 15 (12), 747-756 (2015).</li></ol>

The author has nothing to disclose.

Phospho flow cytometry is a powerful technique to measure protein phosphorylation levels in single cells. Since the method relies on staining with antibodies, phospho flow cytometry is limited by antibody availability. Furthermore, in order to obtain reliable results, all antibodies should be titrated and verified before use. A detailed protocol for titration of phospho-specific antibodies has been described elsewhere12. During panel design, consideration of the signal-to-noise ratio is critical. In the presented example, all phospho-antibodies were conjugated to Alexa Fluor 647. This fluorophore often provides the optimal differential between samples with low versus high levels of phospho-protein. Furthermore, by using only one color for the phospho-proteins the other channels will be left free for FCB and surface marker staining. This panel design reduces spillover into the phospho channel. By having all phospho-antibodies conjugated to the same fluorophore, the data analysis will also be simplified.
In the presented protocol, all antibody stainings were performed after fixation and permeabilization of the cells. However, it is important to keep in mind that surface marker staining can be adversely affected by the fixation and permeabilization steps due to denaturation of the surface antigen or increased nonspecific staining13. The user should therefore test the reactivity of the antibodies on a case to case basis. Resources on compatible clones may also be helpful, such as the overview of different fixation/permeabilization procedures and their compatibility with various antibodies at https://www.cytobank.org/facselect/.
Protein phosphorylation or de-phosphorylation is a transient modification that occurs in response to both extrinsic and intrinsic cues. When comparing phosphorylation patterns, it is therefore crucial that the experiments are carried out under similar conditions. When studying signaling in primary cells from blood, factors that could impact the result include time elapsed after drawing the blood, storage conditions and for how long the isolated cells are rested before initiation of the experiment. When comparing signaling patterns in cryo preserved cells and freshly isolated cells from blood, only very minor significant differences could be observed (Sk&#229;nland, unpublished). However, it is still advisable to use cryo preserved normal cells as a control when studying biobanked patient samples, for example. The optimal conditions for performing the phospho flow cytometry experiments and the impact of external factors should be tested by the individual user.
Here, a protocol is presented for phospho flow analysis of suspension cells. The protocol can be adapted to other cell types, but it is a prerequisite that the cells are in suspension as single cells for the analysis by flow cytometry. The procedure to achieve this must be delicate to preserve, and not affect, phosphorylation patterns. Examples exist where adherent cells are detached from the culturing dish by cold trypsination12,14, or are rather grown on microspheres15. When it comes to phospho flow cytometry on solid tissue, one report exists on lung tumors where single cells were obtained by passing the cells through a tube with a cell strainer16. Recently, phospho flow cytometry was combined with a novel approach termed Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue (DISSECT) in order to study phospho-proteins in epithelial tissues17 and colorectal cancer18.
The FCB is a critical step in the protocol since deconvolution of the samples at the end of the experiment relies on distinct FCB populations. In order to obtain this, the cells need to be homogeneously stained. It is therefore important to prepare a barcoding plate that the cells can be added to. Adding the reagents to the cells will result in uneven staining and mixed populations that cannot be deconvoluted by gating. It is highly recommended to run a test of the barcoding dilutions before the experiment is performed as the staining intensity is cell-type dependent.
Additional antibody-based techniques such as protein array and reverse phase protein array (RPPA) can be applied for quantification of phospho-protein levels in a medium to high-throughput manner. However, some qualities of phospho flow cytometry distinguish this method from the others. An important advantage of phospho flow cytometry is that it allows for single cell profiling. By including surface markers for different cellular subsets, inter-cellular heterogeneity can be detected. Combination with FCB furthermore allows for analysis of several conditions in the same experimental run. These features make phospho flow cytometry an attractive method for future applications in biomarker discovery and precision medicine19.

Phospho flow cytometry is used to analyze protein phosphorylation levels at single-cell resolution. The overall goal of the method is to map cellular signaling patterns under specified conditions. By exploiting the multiparameter capacity of flow cytometry, several signaling pathways can be analyzed simultaneously in different subsets of a heterogeneous cell population such as peripheral blood. These traits offer advantages over other antibody-based technologies such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), protein array, and reverse phase protein array (RPPA)1. Phospho flow cytometry can be combined with fluorescent cell barcoding (FCB), which means that individual cell samples are labeled with unique signatures of fluorescent dyes so that they can be mixed together, stained and analyzed as a single sample2. This reduces the antibody consumption, increases the data robustness through the combination of control and treated samples, and enhances the speed of acquisition. The combined FCB population can then be divided into smaller samples and stained with up to 35 distinct phospho-specific antibodies, depending on the amount of starting material. Large profiling experiments can, thereby, be run with standard cytometer hardware. Phospho flow cytometry has been applied to profile signaling pathways in patient samples from several hematological cancers including chronic lymphocytic leukemia (CLL)3,4,5, acute myeloid leukemia (AML)6 and non-Hodgkin lymphomas7. Phospho flow cytometry is thus a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.
Here, the optimized protocol for analysis of CLL patient samples by phospho flow cytometry is provided (Figure 1A). Examples of basal signaling characterization, anti-IgM/B cell receptor stimulation and drug perturbation are shown. A detailed description of an FCB matrix is provided. The protocol can easily be adapted to other suspension cell types.

<table><tbody><tr><td>RPMI 1640 GlutaMAX</td><td>ThermoFisher Scientific</td><td>61870-010</td><td>Cell culture medium</td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher Scientific</td><td>10270169</td><td>Additive to cell culture medium</td></tr><tr><td>Sodium pyruvate</td><td>ThermoFisher Scientific</td><td>11360-039</td><td>Additive to cell culture medium</td></tr><tr><td>MEM non-essential amino acids</td><td>ThermoFisher Scientific</td><td>11140-035</td><td>Additive to cell culture medium</td></tr><tr><td>Lymphoprep</td><td>Alere Technologies AS</td><td>1114547</td><td>Density gradient medium</td></tr><tr><td>Anti-IgM</td><td>Southern Biotech</td><td>2022-01</td><td>For stimulation of the B cell receptor</td></tr><tr><td>BD Phosflow Fix Buffer I</td><td>BD</td><td>557870</td><td>Fixation buffer</td></tr><tr><td>BD Phosflow Perm Buffer III</td><td>BD</td><td>558050</td><td>Permeabilization buffer</td></tr><tr><td>Alexa Fluor 488 5-TFP</td><td>ThermoFisher Scientific</td><td>A30005</td><td>Barcoding reagent</td></tr><tr><td>Pacific Blue Succinimidyl Ester</td><td>ThermoFisher Scientific</td><td>P10163</td><td>Barcoding reagent</td></tr><tr><td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td><td>ThermoFisher Scientific</td><td>P30253</td><td>Barcoding reagent</td></tr><tr><td>Compensation beads</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td>Correct species reactivity</td></tr><tr><td>Falcon tubes</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td></td></tr><tr><td>Eppendorf tubes</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td></td></tr><tr><td>96 well V-bottom plates</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td>Compatible with the flow cytometer</td></tr><tr><td>Centrifuges</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td>For Eppendorf tubes, Falcon tubes and plates</td></tr><tr><td>Water bath</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td>Temperature regulated</td></tr><tr><td>Flow cytometer</td><td>&lt;em&gt;Defined by user&lt;/em&gt;</td><td></td><td>With High Throughput Sampler (HTS)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AKT (pS473)</td><td>Cell Signaling Technologies</td><td>4075</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; D9E&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce&amp;hellip;, Haematologica, 101(2):e59-62&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9</td></tr><tr><td>ATF-2 (pT71)</td><td>Santa Cruz Biotechnology</td><td>sc-8398</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; F-1&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>BLNK (pY84)</td><td>Beckton Dickinson Pharmingen</td><td>558443</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; J117-1278&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce&amp;hellip;, Haematologica, 101(2):e59-62&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9&lt;br/&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>Btk (pY223)/Itk (pY180)</td><td>Beckton Dickinson Pharmingen</td><td>564846</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; N35-86&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>Btk (pY551)</td><td>Beckton Dickinson Pharmingen</td><td>558129</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 24a/BTK (Y551)&amp;nbsp;&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9</td></tr><tr><td>Btk (pY551)/Itk (pY511)</td><td>Beckton Dickinson Pharmingen</td><td>558134</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 24a/BTK (Y551)&amp;nbsp;&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce&amp;hellip;, Haematologica, 101(2):e59-62</td></tr><tr><td>CD3&amp;zeta; (pY142)</td><td>Beckton Dickinson Pharmingen</td><td>558489</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; K25-407.69&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410</td></tr><tr><td>Histone H3 (pS10)</td><td>Cell Signaling Technologies</td><td>9716</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; D2C8&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td></tr><tr><td>I&amp;kappa;B&amp;alpha;</td><td>Cell Signaling Technologies</td><td>5743</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; L35A5&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>LAT (pY171)</td><td>Beckton Dickinson Pharmingen</td><td>558518</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; I58-1169&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410</td></tr><tr><td>Lck (pY505)</td><td>Beckton Dickinson Pharmingen</td><td>558577</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 4/LCK-Y505&amp;nbsp;&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td></tr><tr><td>MEK1 (pS298)</td><td>Beckton Dickinson Pharmingen</td><td>560043</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; J114-64&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410</td></tr><tr><td>NF-&amp;kappa;B p65 (pS529)</td><td>Beckton Dickinson Pharmingen</td><td>558422</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; K10-895.12.50&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>NF-&amp;kappa;B p65 (pS536)</td><td>Cell Signaling Technologies</td><td>4887</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 93H1&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9</td></tr><tr><td>p38 MAPK (pT180/Y182)</td><td>Cell Signaling Technologies</td><td>4552</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 28B10&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>p44/42 MAPK (pT202/Y204)</td><td>Cell Signaling Technologies</td><td>4375</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; E10&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce&amp;hellip;, Haematologica, 101(2):e59-62&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>p53 (pS15)</td><td>Cell Signaling Technologies</td><td>NN</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 16G8&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&amp;hellip;, Blood, 109(6):2589-96</td></tr><tr><td>p53 (pS20)</td><td>Cell Signaling Technologies</td><td>NN</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; Polyclonal&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&amp;hellip;, Blood, 109(6):2589-96</td></tr><tr><td>p53 (pS37)</td><td>Cell Signaling Technologies</td><td>NN</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; Polyclonal&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&amp;hellip;, Blood, 109(6):2589-96</td></tr><tr><td>p53 (pS46)</td><td>Cell Signaling Technologies</td><td>NN</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; Polyclonal&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&amp;hellip;, Blood, 109(6):2589-96</td></tr><tr><td>p53 (pS392)</td><td>Cell Signaling Technologies</td><td>NN</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; Polyclonal&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&amp;hellip;, Blood, 109(6):2589-96</td></tr><tr><td>PLC&amp;gamma;2 (pY759)</td><td>Beckton Dickinson Pharmingen</td><td>558498</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; K86-689.37&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>Rb (pS807/pS811)</td><td>Beckton Dickinson Pharmingen</td><td>558590</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; J112-906&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>S6-Ribos. Prot. (pS235/236)</td><td>Cell Signaling Technologies</td><td>4851</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; D57.2.2E&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td></tr><tr><td>SAPK/JNK (pT183/Y185)</td><td>Cell Signaling Technologies</td><td>9257</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; G9&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&amp;hellip;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td></tr><tr><td>SLP76 (pY128)</td><td>Beckton Dickinson Pharmingen</td><td>558438</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; J141-668.36.58&amp;nbsp;&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410</td></tr><tr><td>STAT1 (pY701)</td><td>Beckton Dickinson Pharmingen</td><td>612597</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 4a&amp;nbsp;&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>STAT3 (pY705)</td><td>Beckton Dickinson Pharmingen</td><td>557815</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 4/P-STAT3&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td></tr><tr><td>STAT4 (pY693)</td><td>Zymed/ThermoFisher Scientific</td><td>71-7900</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; Polyclonal&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Uzel et al., 2001, Detection of intracellular phosphorylated STAT-4 by flow cytometry, Clin Immunol, 100(3): 270-6</td></tr><tr><td>STAT5 (pY694)</td><td>Beckton Dickinson Pharmingen</td><td>612599</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 47/Stat5(pY694)&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr><tr><td>STAT6 (pY641)</td><td>Beckton Dickinson Pharmingen</td><td>612601</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 18/P-Stat6&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td></tr><tr><td>SYK (pY525/Y526)</td><td>Cell Signaling Technologies</td><td>12081</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; C87C1&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284&lt;br/&gt; Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce&amp;hellip;, Haematologica, 101(2):e59-62</td></tr><tr><td>ZAP70/SYK (pY319/Y352)</td><td>Beckton Dickinson Pharmingen</td><td>557817</td><td>&lt;strong&gt;Clone:&lt;/strong&gt; 17A/P-ZAP70&lt;br/&gt; &lt;strong&gt;Reference:&lt;/strong&gt; Sk&amp;aring;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&amp;hellip;, Biochem J, 460(3):399-410&lt;br/&gt; Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&amp;hellip;, Exp Cell Res, 318(14):1611-9&lt;br/&gt; Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&amp;hellip;, Blood, 129(6): 759-770</td></tr></tbody></table>

phospho flow cytometry with fluorescent cell barcoding for single cell signaling analysis and biomarker discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

The main steps of the phospho flow cytometry protocol are illustrated in Figure 1A. In the presented example, CLL cells were stained with the barcoding reagent Pacific Blue at four dilutions. Three-dimensional barcoding can be performed by combining three barcoding dyes, as illustrated in Figure 1B. The individual samples are then deconvoluted by subsequent gating on each barcoding reagent versus SSC-A (Figure 1C). Detailed information about the barcoding reagents are listed in Table 1.
Following the procedure described here, phospho-protein levels were characterized in B cells from CLL patients and normal controls under various conditions3. Both basal and stimulation-induced phosphorylation levels of 20 signaling molecules downstream of the B cell receptor (BCR) were analyzed (see Table of Materials for a list of reported phospho-specific antibodies). Basal phospho-protein levels were mapped in 22 CLL patient samples relative to the mean of normal controls. This analysis showed that STAT3 (pY705) is significantly upregulated in CLL cells (Figure 1D). Constitutive activation of STAT3 has been reported in other hematological malignancies and is associated with resistance to apoptosis9.
In order to identify signaling aberrations induced through the BCR pathway, cells were stimulated with anti-IgM for up to 30 min. It has been shown that CLL cells from patients with IgVH unmutated status (UM-CLL) display increased sensitivity towards anti-IgM stimulation10. This was indeed observed for the majority of the analyzed proteins, but the effect was statistically significant only for AKT (pS473) (Figure 1E, UM-CLL versus M-CLL and Normal). To test if the aberrant AKT (pS473) signal could be reversed CLL cells were exposed to the PI3K&#948; inhibitor idelalisib, which is used in the clinic to treat CLL patients11. As shown in Figure 1F, AKT (pS473) levels were significantly reduced upon idelalisib treatment in a concentration-dependent manner, demonstrating that kinase inhibitors can be applied to normalize aberrant signaling in CLL cells.
These results show that phospho flow cytometry in combination with FCB is a powerful approach to perform signaling analysis studies, identify potential biomarkers, and assess pharmacodynamics.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58386/58386fig1.jpg" /> 
Figure 1. Work flow and examples of applied phospho flow cytometry analysis. 
(A) The main steps of the phospho flow procedure are illustrated. Cells are first stimulated, then fixed and subjected to FCB before they can be combined in one tube for permeabilization and subsequent antibody staining. The cells are run on a flow cytometer and the cell populations are deconvoluted by gating during the data analysis. The results can be visualized as histograms or heatmaps, as shown. (B) Example of a three-dimensional FCB staining matrix using Alexa Fluor 488 (three dilutions), Pacific Blue (four dilutions) and Pacific Orange (three dilutions). This matrix will allow combination of up to 36 samples. (C) The FCB cell population can be deconvoluted by gating on each FCB channel versus SSC-A. Combination of the gates in the analysis software generates the correct populations for analysis. (D) Unstimulated B cells from healthy donors (n = 25) and CLL patients (n = 22) were subjected to analysis by phospho flow following the procedure in (A). The basal fluorescence intensity signals were calculated relative to IgG&#954; isotype control as arcsinh ratio. The signals in CLL B cells were then normalized to the signals in B cells from normal controls. **p &#60; 0.01, calculated by an unpaired two-sample t-test. UM-CLL: IgVH unmutated CLL, M-CLL: IgVH mutated CLL. Symbols of the same color represent patient samples which grouped together in a hierarchical agglomerative cluster based on levels of 20 phospho-proteins3. (E) B cells from normal controls (n = 10, mean + SEM) or CLL patients (n = 11 [M-CLL] and n = 8 [UM-CLL], mean + SEM) were stimulated with anti-IgM for the indicated time-course and subjected to phospho flow analysis. The fluorescence intensity signals were measured relative to unstimulated samples and shown as arcsinh ratio. **p &#60; 0.01 (Normal vs UM-CLL) and ***p &#60; 0.001 (M-CLL vs UM-CLL), calculated by multiple comparison testing with Holm-Sidak&#39;s correction. UM-CLL: IgVH unmutated CLL, M-CLL: IgVH mutated CLL. (F) CLL cells were incubated with DMSO or idelalisib as indicated for 20 min before anti-IgM stimulation for 3 min. The cells were then processed following the phospho flow protocol. *p &#60; 0.05, **p &#60; 0.01, ****p &#60; 0.0001, calculated by multiple comparison testing with Holm-Sidak&#39;s correction. UM-CLL: IgVH unmutated CLL, M-CLL: IgVH mutated CLL. See (D) for explanation of symbol color. (D-F) are modified from3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58386/58386fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="5">Serial dilute as follows (starting with the stock solution)</td>
		</tr>
		<tr>
			<td>Barcoding reagent</td>
			<td>Stock concentration</td>
			<td>#1</td>
			<td>#2</td>
			<td>#3</td>
			<td>#4</td>
			<td>unstained</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>10 mg/mL</td>
			<td>1:500</td>
			<td>1:5</td>
			<td></td>
			<td></td>
			<td>x</td>
		</tr>
		<tr>
			<td>Pacific Blue</td>
			<td>10 mg/mL</td>
			<td>1:2500</td>
			<td>1:4</td>
			<td>1:4</td>
			<td>1:10</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange</td>
			<td>2 mg/mL</td>
			<td>1:50</td>
			<td>1:12</td>
			<td>1:24</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Barcoding reagents.

Watch this Scientific Journal Video about Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery at JoVE.com

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

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Unit 1

Principles of Cell Signaling

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21.2K Views.  University of Oslo. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van celbiologie en immunologie, zoals hoe worden signaleringstrajecten beïnvloed door verschillende stimuli of medicijnen? Het belangrijkste voordeel van deze techniek is dat het u toelaat om eencellige signalering analyse uit te voeren in de medium tot hoge doorvoer mode. Na het opnieuw ophangen van de cellen in het aanvullende RPMI 16/40-medium, breng je de vereiste hoeveelheid celsuspensie over naar de putte...

Video: Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

Biochemistry

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Immunology and Infection

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Biology

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Source: Baxter, R. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57780">Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.</a> J. Vis. Exp. (2018).
This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells.

1. Barcode of Lysed/Fixed Blood Cells

	Thaw the lysed/fixed cell samples from the -80 &#176;C storage slowly on ice; a maximum of 20 samples can be ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

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