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 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.

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) from CLL Patient Blood Samples

CAUTION: Human blood should be handled according to regulations for Biosafety Level 2.

1. 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).
2. Carefully layer 10 mL of a density gradient medium (e.g., Lymphoprep) to the bottom of the tube using a 10 mL pipette.
3. Centrifuge at 800 x g for 20 min at 4 °C. The PBMCs are now visible on top of the density gradient medium layer.
4. Use a Pasteur pipette to transfer the cells into two new 50 mL tubes. Wash twice with PBS (fill up the tubes).
5. Centrifuge at 350 x g for 15 min. Discard the supernatant and resuspend in 3 mL of PBS.
6. Count the cells using a preferred method.
7. Centrifuge the cells at 350 x g for 5 min. Discard the supernatant. NOTE: Steps 1.8 to 3.2 are optional. It is possible to proceed directly to step 3.3.
8. 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.

2. Thawing of Cells

1. Quickly thaw the cells in a 37 °C water bath.
   NOTE: DMSO is toxic to the cells. Work fast to limit the exposure to DMSO.
2. Wash the cells once with 10 mL of cold Roswell Park Memorial Institute medium (RPMI 1640 with supplemental GlutaMAX, see Table of Materials).
3. Centrifuge at 300 x g for 5 min. Discard the supernatant.
4. 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 °C for 1 hour to allow the cells to calibrate.

3. Preparation of Cells

1. Count the viable cells using a preferred method.
2. Transfer the cells to a 50 mL tube and centrifuge at 300 x g for 5 min. Discard the supernatant.
3. Resuspend the cells in RPMI 1640 medium (step 2.2) supplemented with 1% FBS to no more than 50 x 10⁶ cells/mL.
4. 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 µL sample per time-point + 50 µL of dead volume. Save samples for compensation controls (one unstained sample + one sample per barcoding dye).
5. Transfer the 96 well plate to a pre-heated 37 °C water bath. Rest the cells for 10 min.

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.

1. Prepare a 96 well V-bottom plate with 60 µL of Fix Buffer I per well per sample. Leave in the 37 °C water bath.
   NOTE: Cells: Fix buffer should be 1:1. In order to allow for evaporation at 37 °C, the Fix buffer is initially in abundance.
2. Optionally, treat the cells with drugs before stimulation.
3. Transfer a 50 µL control sample to the fix plate. Mix by pipetting up and down.
4. Optionally, start the stimulation time-course by adding 10 µg/mL anti-IgM to the cells. Mix by pipetting up and down.
5. Transfer a 50 µL 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).
6. Leave the fix plate at 37 °C for 10 min after the last sample has been added.

5. Fluorescent Cell Barcoding (FCB)

NOTE: See Table 1 for a list of barcoding reagents.

1. Wash the fixed cells 3x with PBS (fill up the wells).
2. Centrifuge at 500 x g for 5 min. Discard the supernatant.
3. Prepare a 96 well V-bottom plate with barcoding reagents. Pipet 5 µL 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.
4. Resuspend the cells in 190 µL 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.
5. Leave the cells for 20 min at room temperature, in the dark.
6. Wash the stained cells 2x with flow wash (PBS, 1% FBS, 0.09% sodium azide) (fill up the wells).
7. Centrifuge at 500 x g for 5 min. Discard the supernatant.
8. Add 190 µL 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.
9. Centrifuge at 500 x g for 5 min. Discard the supernatant.

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.

1. Transfer 2 mL of Perm Buffer III to a 15 mL tube. Leave at -20 °C so it is ice-cold upon use.
   NOTE: The Perm Buffer can be left at -20 °C from the start of the experiment.
2. Add 1.5 mL of ice-cold Perm Buffer to the barcoded cell population (in a 15 mL tube) and 100 µL to each compensation control (in 1.7 mL tubes) drop-wise while vortexing to avoid that the cells clump together.
3. Transfer the cells directly to -80 °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 °C.

7. Antibody Staining

NOTE: See Table of Materials for a list of reported phospho-specific antibodies.

1. Transfer the cells from -80 °C to a box of ice.
2. 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.
3. Centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
4. Resuspend the barcoded cell population in a volume of flow wash, which allows 25 µL of cell suspension per phospho-antibody stain. Resuspend the compensation controls in 200 µL of flow wash.
5. Prepare antibodies for staining in a 96 well V-bottom plate. The final volume will be 50 µL/well. Per well, add phospho-specific antibody diluted in flow wash to a final volume of 10 µL, surface marker diluted in flow wash to a final volume of 15 µL, and 25 µL of cell suspension.
   NOTE: Antibody dilutions should be titrated prior to the experiment. Include isotype control.
6. Leave the cells for 30 min at room temperature, in the dark.
7. Wash the stained cells 2x with flow wash (fill up the wells).
8. Centrifuge at 500 x g for 5 min. Discard the supernatant.
9. Resuspend the cells in 150 µL of flow wash.

8. Preparation of Compensation Controls

1. Prepare compensation controls for the antibody-conjugated fluorochromes in parallel with the antibody staining. Use compensation beads according to the vendor’s instructions.

9. Flow Cytometry Analysis

NOTE: The experiment can be run on a flow cytometer with a High Throughput Sampler (HTS).

1. Optimize the photomultiplier tube (PMT) voltage with the unstained control.
2. Run compensation controls and calculate the compensation matrix.
3. Run samples. The event rate should be in accordance with the instrument specifications.

10. Gating Strategy and Data Analysis

1. Import the FCS files from the experiment to a flow cytometry analysis software like FlowJo or Cytobank (https://cellmass.cytobank.org).
2. Gating strategy
   1. Select lymphocytes by plotting SSC-A versus FSC-A in a density dot plot.
   2. Display the lymphocytes and select the singlets by plotting SSC-A versus FSC -W.
   3. Display the single cells and gate the cell type by plotting SSC-A versus the surface marker.
   4. 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).
   5. Plot the phospho antibody channel against the FCB channel, or as a heatmap (see Figure 1A) to display the phosphorylation events.
3. 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).

Results

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 (

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

List of materials used in this article

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