evaluation of stromal cytokine-induced intracellular protein phosphorylation using phospho-flow cytometry

Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

To validate the supernatant produced from engineered stroma, thaw the supernatant samples harvested from the control in cytokine-expressing stroma, and plate 3 wells of leukemia cell lines per condition in R20 medium at a 2 times 10 to the fifth cells per well concentration in a 96-well tissue culture plate. Incubate the cells for two hours at 37 degrees Celsius to allow for the phosphorylation from their prior culture to be lost.
Then, transfer the cells from each well into individual 4-milliliter tubes, and collect the cells by centrifugation. Resuspend the pellets in 600 microliters per experimental condition, and plate 200 microliters of cells per well for each condition in triplicate. Incubate the cells for the appropriate period according to the specific commercial phospho-assay.
At the end of the incubation, pellet the cells by centrifugation, and label the cells via phospho-flow cytometry staining according to the manufacturer&#39;s protocol. Then, analyze the cells by flow cytometry using standard flow cytometric analysis protocols to verify that cytokine in the stroma supernatant phosphorylates the leukemia cells.

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1. Phospho-flow Cytometry Assays to Evaluate the Activity of Cytokine Produced by Stroma
NOTE: Assess the supernatant from the engineered stroma before the first experiment to verify the relevant bio-activity of the stroma-generated cytokine for the individual experiment. Once the engineered stroma is validated, further testing is not necessary unless stroma are introduced that were produced with a new vector.
<ol>
	<li>Purchase MUTZ5 and/or MHH-CALL4 leukemia cell lines (TSLP responsive), recombinant human cytokine, and antibodies approved for use in phospho-flow cytometry assays to detect appropriate downstream phosphorylation targets.</li>
	<li>Thaw harvested supernatant from control and cytokine-expressing stroma.</li>
	<li>Plate the leukemia cell lines in R20 medium (see Table of Materials) at a concentration of 0.2 x 106 cells per well in a 96-well tissue culture plate. Plate 3 wells per condition to allow for triplicate assays. Incubate for 2 h at 37 &#176;C. This time allows for the loss of any phosphorylation that occurred during prior culture conditions. 
	NOTE: 12 wells per cell line provides triplicate assays for each experimental condition shown in step 1.4.</li>
	<li>After 2 h in culture, transfer the cells from each well to separate 4 mL tubes and centrifuge at 500 x g for 5 min, at 4 &#176;C. Decant the supernatant.
	<ol>
		<li>Resuspend the cells in 200 &#181;L for each of the following experimental conditions (perform assays in triplicate): 1) negative control-media only, 2) positive control-media with recombinant cytokine at saturating concentration(s) (TSLP at 15 ng/mL, etc.), 3) control stroma-supernatant from control stroma, and 4) cytokine-stroma-supernatant from cytokine-producing stroma.</li>
	</ol>
	</li>
	<li>Incubate the cells for the duration specified by specific commercial phospho-assay (e.g. 30 min for phospho-STAT5 in MUTZ5 or MHH-CALL4 in response to TSLP).</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C and decant the supernatant.</li>
	<li>Perform phospho-flow cytometry staining per the manufacturer&#39;s protocol and collect flow cytometry data.</li>
	<li>Analyze flow cytometry data.
	<ol>
		<li>Gate on intact cells based on forward (FSC, indicates cell size) and side (SSC, indicates cell granularity) light scatter.</li>
		<li>Determine the median fluorescent intensity (MFI) of the intact cells in each sample. Compare the average MFI obtained from the triplicate values of stromal cell supernatant to that of positive and negative media controls.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene conical tubes</td>
			<td>Fisher</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipette tips</td>
			<td>Fisher</td>
			<td>2707509</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Denville Scientific</td>
			<td>P3020-CPS</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L pipette tips</td>
			<td>Denville Scientific</td>
			<td>P-1096-CP</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;R20&#8221; cell culture medium, % of total volume (makes 195 mL)</td>
			<td></td>
			<td></td>
			<td>Lab Recipe</td>
		</tr>
		<tr>
			<td>RPMI, 76.84% (150 mL)</td>
			<td>Mediatech</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, 20.49% (40 mL)</td>
			<td>Mediatech</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>2mM L-glutamine, 1.02% (2 mL)</td>
			<td>Mediatech</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mM Na pyruvate, 1.02% (2 mL )</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, 0.51% (1 mL</td>
			<td>Mediatech</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol, 0.10% (0.1 M), 0.10% (200 &#181;L)</td>
			<td>MP</td>
			<td>190242</td>
			<td></td>
		</tr>
		<tr>
			<td>Biologics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;CALL-4&#34; MHH-CALL-4 cell line, human B cell precursor leukemia</td>
			<td>DSMZ</td>
			<td>ACC 337</td>
			<td></td>
		</tr>
		<tr>
			<td>MUTZ-5 cell line, human B cell precursor leukemia</td>
			<td>DSMZ</td>
			<td>ACC 490</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TSLP protein</td>
			<td>R&#38;D Systems</td>
			<td>1398-TS-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytomery antibodies (clone)*</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 FITC (30F11)</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-778</td>
			<td></td>
		</tr>
		<tr>
			<td>CD127 PE (MB15-18C9) (alternate name is IL-7R&#945;)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-094</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 APC (8G12)</td>
			<td>BD Biosciences</td>
			<td>340667</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 PE-Cy7 (HI30)</td>
			<td>eBioscience</td>
			<td>25-0459</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;FVD&#34; Fixable viability dye eFluor 780</td>
			<td>eBioscience</td>
			<td>65-0865</td>
			<td></td>
		</tr>
		<tr>
			<td>Ig &#954; light chain FITC (G20-193)</td>
			<td>BD Pharmingen</td>
			<td>555791</td>
			<td></td>
		</tr>
		<tr>
			<td>Ig &#955; light chain FITC (JDC-12)</td>
			<td>BD Pharmingen</td>
			<td>561379</td>
			<td></td>
		</tr>
		<tr>
			<td>IgD PE (IA6-2)</td>
			<td>BioLegend</td>
			<td>348203</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM PE-Cy5 (G20-127)</td>
			<td>BD Biosciences</td>
			<td>551079</td>
			<td></td>
		</tr>
		<tr>
			<td>pSTAT5 PE, mouse anti-STAT5 (pY694)</td>
			<td>BD PhosphoFlow</td>
			<td>612567</td>
			<td></td>
		</tr>
		<tr>
			<td>Other materials &#38; equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo flow cytometry analysis software</td>
			<td>FlowJo, LLC</td>
			<td>Version 10</td>
			<td></td>
		</tr>
		<tr>
			<td>*All antibodies are anti-human unless otherwise stated.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Coats, J. S., et al. <a href="https://app.jove.com/v/55384">Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line.</a> J. Vis. Exp. (123) (2017).
This video demonstrates the evaluation of stromal cytokine-induced intracellular phosphorylation of proteins in leukemia cells. These intracellular phosphorylated proteins are quantified using the phospho-flow cytometry technique.

1. Phospho-flow Cytometry Assays to Evaluate the Activity of Cytokine Produced by Stroma
NOTE: Assess the supernatant from the engineered stroma before ...

Take leukemia cells in growth media without stimulating factors such as cytokines, and incubate them.
The absence of stimulating factors allows intracellular phosphorylated proteins to undergo dephosphorylation and return to their native states.
Centrifuge and remove the media. Resuspend cells in stroma-supernatant containing the cytokine, thymic stromal lymphopoietin, or TSLP.
TSLP binds to its receptor on the leukemia cell, triggering receptor dimerization.
This brings the two Janus-associated kinases, or JAKs, closer, facilitating mutual transphosphorylation .
The activated JAKs phosphorylate the cytoplasmic tails of the receptor, creating docking sites for signal transducer and activator of transcription, STAT monomers.
Once phosphorylated, STATs dimerize and detach from the receptor to regulate downstream cellular processes.
Fix the cells to maintain the phosphorylation states of intracellular proteins.
Treat the cells with a permeabilization solution to increase the cell membrane permeability.
Add fluorescently labeled antibodies to label the phosphorylated proteins.
Flow-cytometric measurement of fluorescence signal correlates with TSLP-induced STAT phosphorylation.

55 Views. Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

Video: Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry - Experiment

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.

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

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Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

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Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

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Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry