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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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).......

Discussion

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. .......

Materials

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