Name: phospho flow cytometry with fluorescent cell barcoding for single cell signaling analysis and biomarker discovery
Uploaded: 2018-10-04T14:45:00
Description: Watch this Scientific Journal Video about Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery at JoVE.com

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)...

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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. ...

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>Defined by user</td>
			<td></td>
			<td>Correct species reactivity</td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Defined by user</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Defined by user</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-bottom plates</td>
			<td>Defined by user</td>
			<td></td>
			<td>Compatible with the flow cytometer</td>
		</tr>
		<tr>
			<td>Centrifuges</td>
			<td>Defined by user</td>
			<td></td>
			<td>For Eppendorf tubes, Falcon tubes and plates</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Defined by user</td>
			<td></td>
			<td>Temperature regulated</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Defined by user</td>
			<td></td>
			<td>With High Throughput Sampler (HTS)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antigen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKT (pS473)</td>
			<td>Cell Signaling Technologies</td>
			<td>4075</td>
			<td>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&#8230;, Haematologica, 101(2):e59-62 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, 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>Clone: F-1 
			Reference: Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&#8230;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td>
		</tr>
		<tr>
			<td>BLNK (pY84)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558443</td>
			<td>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&#8230;, Haematologica, 101(2):e59-62 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, Exp Cell Res, 318(14):1611-9 
			Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>Btk (pY223)/Itk (pY180)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>564846</td>
			<td>Clone: N35-86 
			Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>Btk (pY551)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558129</td>
			<td>Clone: 24a/BTK (Y551)&#160; 
			Reference: Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, 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>Clone: 24a/BTK (Y551)&#160; 
			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&#8230;, Haematologica, 101(2):e59-62</td>
		</tr>
		<tr>
			<td>CD3&#950; (pY142)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558489</td>
			<td>Clone: K25-407.69 
			Reference: Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410</td>
		</tr>
		<tr>
			<td>Histone H3 (pS10)</td>
			<td>Cell Signaling Technologies</td>
			<td>9716</td>
			<td>Clone: D2C8 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284</td>
		</tr>
		<tr>
			<td>I&#954;B&#945;</td>
			<td>Cell Signaling Technologies</td>
			<td>5743</td>
			<td>Clone: L35A5 
			Reference: Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>LAT (pY171)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558518</td>
			<td>Clone: I58-1169 
			Reference: Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410</td>
		</tr>
		<tr>
			<td>Lck (pY505)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558577</td>
			<td>Clone: 4/LCK-Y505&#160; 
			Reference: 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>Clone: J114-64 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410</td>
		</tr>
		<tr>
			<td>NF-&#954;B p65 (pS529)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558422</td>
			<td>Clone: K10-895.12.50 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, Exp Cell Res, 318(14):1611-9 
			Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&#8230;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td>
		</tr>
		<tr>
			<td>NF-&#954;B p65 (pS536)</td>
			<td>Cell Signaling Technologies</td>
			<td>4887</td>
			<td>Clone: 93H1 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, 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>Clone: 28B10 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&#8230;, 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>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&#8230;, Haematologica, 101(2):e59-62 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, Exp Cell Res, 318(14):1611-9 
			Pollheimer et al., 2013, Interleukin-33 drives a proinflammatory endothelial&#8230;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td>
		</tr>
		<tr>
			<td>p53 (pS15)</td>
			<td>Cell Signaling Technologies</td>
			<td>NN</td>
			<td>Clone: 16G8 
			Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&#8230;, Blood, 109(6):2589-96</td>
		</tr>
		<tr>
			<td>p53 (pS20)</td>
			<td>Cell Signaling Technologies</td>
			<td>NN</td>
			<td>Clone: Polyclonal 
			Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&#8230;, Blood, 109(6):2589-96</td>
		</tr>
		<tr>
			<td>p53 (pS37)</td>
			<td>Cell Signaling Technologies</td>
			<td>NN</td>
			<td>Clone: Polyclonal 
			Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&#8230;, Blood, 109(6):2589-96</td>
		</tr>
		<tr>
			<td>p53 (pS46)</td>
			<td>Cell Signaling Technologies</td>
			<td>NN</td>
			<td>Clone: Polyclonal 
			Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&#8230;, Blood, 109(6):2589-96</td>
		</tr>
		<tr>
			<td>p53 (pS392)</td>
			<td>Cell Signaling Technologies</td>
			<td>NN</td>
			<td>Clone: Polyclonal 
			Reference: Irish et al., 2007, Flt3 Y591 duplication and Bcl-2 overexpression&#8230;, Blood, 109(6):2589-96</td>
		</tr>
		<tr>
			<td>PLC&#947;2 (pY759)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558498</td>
			<td>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&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>Rb (pS807/pS811)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558590</td>
			<td>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&#8230;, 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>Clone: D57.2.2E 
			Reference: 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>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&#8230;, Arterioscler Thromb Vasc Biol, 33(2):e47-55</td>
		</tr>
		<tr>
			<td>SLP76 (pY128)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>558438</td>
			<td>Clone: J141-668.36.58&#160; 
			Reference: Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410</td>
		</tr>
		<tr>
			<td>STAT1 (pY701)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>612597</td>
			<td>Clone: 4a&#160; 
			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&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>STAT3 (pY705)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>557815</td>
			<td>Clone: 4/P-STAT3 
			Reference: 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>Clone: Polyclonal 
			Reference: 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>Clone: 47/Stat5(pY694) 
			Reference: Myhrvold et al., 2018, Single cell profiling of phospho-protein levels in.., Oncotarget, 9(10):9273-9284 
			Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&#8230;, Blood, 129(6): 759-770</td>
		</tr>
		<tr>
			<td>STAT6 (pY641)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>612601</td>
			<td>Clone: 18/P-Stat6 
			Reference: 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>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&#8230;, Haematologica, 101(2):e59-62</td>
		</tr>
		<tr>
			<td>ZAP70/SYK (pY319/Y352)</td>
			<td>Beckton Dickinson Pharmingen</td>
			<td>557817</td>
			<td>Clone: 17A/P-ZAP70 
			Reference: Sk&#229;nland et al., 2014, T-cell co-stimulation through the CD2 and CD28&#8230;, Biochem J, 460(3):399-410 
			Kalland et al., 2012, Modulation of proximal signaling in normal and transformed&#8230;, Exp Cell Res, 318(14):1611-9 
			Myklebust et al., 2017, Distinct patterns of B-cell receptor signaling in&#8230;, 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 (<strong class="xf...

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 ...

This method can help answer key questions in the fields of cell biology and immunology such as how are signaling pathways affected by different stimuli or drugs? The main advantage of this technique is that it allows you to perform single cell signaling analysis in the medium to high throughput fashion. After re-suspending the cells in supplemented RPMI 16/40 medium, transfer the required amount of cell suspension to the wells of a 96 well V-bottom plate.Transfer the plate to a preheated 37 degree Celsius water bath and allow it to rest there for 10 minutes. Then add 60 microliters of fixed buffer one to each well of a new 96 well plate to set up the fix plate. Place this plate in the 37 degree Celsius water bath.Transfer a 50 microliter control sample to the plate and mix by pipetting up and down. Optionally, add 10 microliters per milliliter anti-IgM to the cells to start the simulation time course and mix by pipetting up and down. Transfer a 50 microliter sample to the fix plate at each time point, mixing each sample as it is added.After the last sample is added, leave the fix plate in the water bath for 10 minutes. First, wash the cells three times with PBS. Centrifuge the plate at 500 times G for five minutes and discard the supernatant.Next, prepare a fresh 96 well V-bottom plate with barcoding reagents, by pipetting five microliters of each barcoding reagent into each well filling the number of wells required to stain each of the samples. In the fixed cell plate, resuspend the cells in each well with 190 microliters of PBS. Then, transfer each well of cells to the corresponding well in the barcoding plate, mixing each well thoroughly.Let the plate rest at room temperature for 20 minutes in the dark. Centrifuge the plate at 500 times G for five minutes and discard the supernatant. After this, wash the cells in each well twice with the flow wash.Then add 190 microliters of flow wash to each well of cells. Combine all of the barcoded samples into one 15 milliliter tube and transfer each compensation control to a separate 1.7 milliliter tube. Centrifuge at 500 times G for five minutes and discard the supernatant.To begin, transfer two milliliters of perm buffer three to a 15 milliliter tube. Store at minus 20 degrees Celsius so it will be ice cold when used. When ready, add 1.5 milliliters of ice cold perm buffer to the 15 milliliter tube containing the barcoded cell population dropwise while vortexing to avoid cell clumping.Add 100 microliters of ice cold perm buffer to each of the compensation controls in the same manner. Immediately transfer the cells to a freezer at minus 80 degrees Celsius for a minimum of 30 minutes. When ready to begin antigen staining, remove the cells from the freezer and transfer them to a box of ice.Wash the cells three times with an excess of flow wash. Centrifuge the cells at 500 times G and four degrees Celsius for five minutes. Discard the supernatant and re-suspend the barcoded cell population in a volume of flow wash, such is that there is 25 microliters of cell suspension per phospho-antibody stain.Re-suspend the compensation controls in 200 microliters of flow wash. To begin preparing antibodies for staining, add 10 microliters of phospho-specific antibody, diluted in flow wash, to each well of a fresh 96 well V-bottom plate. Add 15 microliters of surface marker, diluted in flow wash and 25 microliters of cell suspension to each well for a final volume of 50 microliters per well.Let the cells rest for 30 minutes at room temperature in the dark. After this, wash the stained cells twice with flow wash. Centrifuge at 500 times G for five minutes.Discard the supernatant and re-suspend the cells in 150 microliters of flow wash. Then perform flow cytometry analysis as outlined in the text protocol. In the presented protocol, three dimensional barcoding is performed by combining three barcoding dyes.Individual samples are then de-convoluted by subsequent gating on each barcoding reagent versus side scatter area. The basal and stimulation induced phosphorylation levels of 20 signaling molecules downstream of the B cell receptor are then characterized in B cells from CLL patients and normal controls under various conditions. The stat3 phospho tyrosine 705 is seen to be significantly up regulated.Cells are stimulated with anti-IgM for up to 30 minutes in order to identify signaling aberrations induced through the B cell receptor pathway. While CLL cells from patients with unmutated IGVH display increased sensitivity towards anti-IgM stimulation, it is only statistically significant for AKT phosphor serine 473. Cells are then exposed to the PI 3-kinase delta inhibitor idelalisib to test if the aberrant AKT pS473 signal can be reversed.As shown here, the aberrant levels are significantly reduced upon treatment in a concentration dependent manner demonstrating that kinase inhibitors can be applied to normalize aberrant signaling in CLL cells. Before attempting this procedure it's important to remember to titrate barcoding reagents and antibodies, and to verify that selected surface markers are compatible with fixation and permeabilization reagents. The cells need to be in suspension for phosphor analysis, still the protocol can be adapted to aberrant cells or tissue following procedures to obtain single cell suspension, such as whole trypzination.

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Principles of Cell Signaling

SCIENTISTS IN ACTION

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Cell Cycle-specific Measurement of &amp;#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Far-Red Fluorescent Senescence-Associated &amp;#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

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 ...

&lt;em&gt;TMEM200A&lt;/em&gt;: A Novel Oncogene Regulating EMT and PI3K/AKT in Gastric Cancer

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in common cancers by multiomics analysis and used in vitro cell cultures of gastric cells to verify the results. The expression of TMEM200A in several human cancer types was assessed using the RNA-seq data from the UCSC Xena database. Bioinformatic analysis revealed a potential role of TMEM200A as a diagnostic and prognostic biomarker. 
Cultures of normal gastric and cancer cell lines were grown and TMEM200A was knocked down. The expression levels of TMEM200A were measured by using quantitative real-time polymerase chain reaction and western blotting. In vitro loss-of-function studies were then used to determine the roles of TMEM200A in the malignant behavior and tumor formation of gastric cancer (GC) cells. Western blots were used to assess the effect of the knockdown on epithelial-mesenchymal transition (EMT) and PI3K/AKT signaling pathway in GC. Bioinformatic analysis showed that TMEM200A was expressed at high levels in GC. 
The proliferation of GC cells was inhibited by TMEM200A knockdown, which also decreased vimentin, N-cadherin, and Snai proteins, and inhibited AKT phosphorylation. The PI3K/AKT signaling pathway also appeared to be involved in TMEM200A-mediated regulation of GC development. The results presented here suggest that TMEM200A regulates the tumor microenvironment by affecting the EMT. TMEM200A may also affect EMT through PI3K/AKT signaling, thus influencing the tumor microenvironment. Therefore, in pan-cancers, especially GC, TMEM200A may be a potential biomarker and oncogene.

Author Spotlight: Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies 

Here, a protocol is presented in which multiple bioinformatic tools are combined to study the biological functions of TMEM200A in cancer. In addition, we also experimentally validate the bioinformatics predictions.

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in ...