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Protocol

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Materials

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Cancer Research

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

Published: October 4th, 2018

DOI:

10.3791/58386

1Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, 2K. G. Jebsen Centre for B cell malignancies and K. G. Jebsen Centre for Cancer Immunotherapy, University of Oslo

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 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.

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 ....

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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).......

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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 (

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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. .......

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This work was conducted in the lab of Professor Kjetil Taskén, 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.

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Name Company Catalog Number Comments
RPMI 1640 GlutaMAX ThermoFisher Scientific 61870-010 Cell culture medium
Fetal bovine serum ThermoFisher Scientific 10270169 Additive to cell culture medium
Sodium pyruvate ThermoFisher Scientific 11360-039 Additive to cell culture medium
MEM non-essential amino acids ThermoFisher Scientific 11140-035 Additive to cell culture medium
Lymphoprep Alere Technologies AS 1114547 Density gradient medium
Anti-IgM Southern Biotech 2022-01 For stimulation of the B cell receptor
BD Phosflow Fix Buffer I BD 557870 Fixation buffer
BD Phosflow Perm Buffer III BD 558050 Permeabilization buffer
Alexa Fluor 488 5-TFP ThermoFisher Scientific A30005 Barcoding reagent
Pacific Blue Succinimidyl Ester ThermoFisher Scientific P10163 Barcoding reagent
Pacific Orange Succinimidyl Ester, Triethylammonium Salt ThermoFisher Scientific P30253 Barcoding reagent
Compensation beads Defined by user Correct species reactivity
Falcon tubes Defined by user
Eppendorf tubes Defined by user
96 well V-bottom plates Defined by user Compatible with the flow cytometer
Centrifuges Defined by user For Eppendorf tubes, Falcon tubes and plates
Water bath Defined by user Temperature regulated
Flow cytometer Defined by user With High Throughput Sampler (HTS)
Name Company Catalog Number Comments
Antigen
AKT (pS473) Cell Signaling Technologies 4075 Clone: D9E
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
ATF-2 (pT71) Santa Cruz Biotechnology sc-8398 Clone: F-1
Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
BLNK (pY84) Beckton Dickinson Pharmingen 558443 Clone: J117-1278
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
Btk (pY223)/Itk (pY180) Beckton Dickinson Pharmingen 564846 Clone: N35-86
Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
Btk (pY551) Beckton Dickinson Pharmingen 558129 Clone: 24a/BTK (Y551) 
Reference: Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
Btk (pY551)/Itk (pY511) Beckton Dickinson Pharmingen 558134 Clone: 24a/BTK (Y551) 
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62
CD3ζ (pY142) Beckton Dickinson Pharmingen 558489 Clone: K25-407.69
Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Histone H3 (pS10) Cell Signaling Technologies 9716 Clone: D2C8
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
IκBα Cell Signaling Technologies 5743 Clone: L35A5
Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
LAT (pY171) Beckton Dickinson Pharmingen 558518 Clone: I58-1169
Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Lck (pY505) Beckton Dickinson Pharmingen 558577 Clone: 4/LCK-Y505 
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
MEK1 (pS298) Beckton Dickinson Pharmingen 560043 Clone: J114-64
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
NF-κB p65 (pS529) Beckton Dickinson Pharmingen 558422 Clone: K10-895.12.50
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
NF-κB p65 (pS536) Cell Signaling Technologies 4887 Clone: 93H1
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
p38 MAPK (pT180/Y182) Cell Signaling Technologies 4552 Clone: 28B10
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
p44/42 MAPK (pT202/Y204) Cell Signaling Technologies 4375 Clone: E10
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
p53 (pS15) Cell Signaling Technologies NN Clone: 16G8
Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96
p53 (pS20) Cell Signaling Technologies NN Clone: Polyclonal
Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96
p53 (pS37) Cell Signaling Technologies NN Clone: Polyclonal
Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96
p53 (pS46) Cell Signaling Technologies NN Clone: Polyclonal
Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96
p53 (pS392) Cell Signaling Technologies NN Clone: Polyclonal
Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression…, Blood, 109(6):2589-96
PLCγ2 (pY759) Beckton Dickinson Pharmingen 558498 Clone: K86-689.37
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
Rb (pS807/pS811) Beckton Dickinson Pharmingen 558590 Clone: J112-906
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
S6-Ribos. Prot. (pS235/236) Cell Signaling Technologies 4851 Clone: D57.2.2E
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
SAPK/JNK (pT183/Y185) Cell Signaling Technologies 9257 Clone: G9
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial…, Arterioscler Thromb Vasc Biol, 33(2):e47-55
SLP76 (pY128) Beckton Dickinson Pharmingen 558438 Clone: J141-668.36.58 
Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
STAT1 (pY701) Beckton Dickinson Pharmingen 612597 Clone: 4a 
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
STAT3 (pY705) Beckton Dickinson Pharmingen 557815 Clone: 4/P-STAT3
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
STAT4 (pY693) Zymed/ThermoFisher Scientific 71-7900 Clone: Polyclonal
Reference: Uzel et al., 2001, Detection of intracellular phosphorylated STAT-4 by flow cytometry, Clin Immunol, 100(3): 270-6
STAT5 (pY694) Beckton Dickinson Pharmingen 612599 Clone: 47/Stat5(pY694)
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770
STAT6 (pY641) Beckton Dickinson Pharmingen 612601 Clone: 18/P-Stat6
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
SYK (pY525/Y526) Cell Signaling Technologies 12081 Clone: C87C1
Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284
Parente-Ribes et al., 2016, Spleen tyrosine kinase inhibitors reduce…, Haematologica, 101(2):e59-62
ZAP70/SYK (pY319/Y352) Beckton Dickinson Pharmingen 557817 Clone: 17A/P-ZAP70
Reference: Skånland et al., 2014, T-cell co-stimulation through the CD2 and CD28…, Biochem J, 460(3):399-410
Kalland et al., 2012, Modulation of proximal signaling in normal and transformed…, Exp Cell Res, 318(14):1611-9
Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in…, Blood, 129(6): 759-770

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