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EoE Sub Articles

1.&#160;Preparation of Controls
<ol>
	<li>Transfer the following into separate and appropriately labeled 1.5 ml tubes:
	<ol>
		<li>Add 500 &#181;L of fresh IMDM containing resuspended DiO-labeled K562 cells into the &#34;Double Positive&#34; labeled 1.5 mL tube.</li>
		<li>Add 500 &#181;L of fresh IMDM containing resuspended DiO-labeled K562 cells into the &#34;DiO only&#34; labeled 1.5 mL tube.</li>
		<li>Add 500 &#181;L of fresh IMDM containing resuspended unlabeled K562 cells into the &#34;Propidium Iodide (PI) only&#34; labeled 1.5 mL tube.</li>
	</ol>
	</li>
	<li>Place the Double Positive and PI-only tubes in a 55 &#176;C water bath for 5 min.</li>
	<li>After the 5 min has elapsed, remove the tubes and wipe them down with 70% ethanol.</li>
	<li>Add 10 L of PI to the Double Positive and PI only 1.5 mL tubes.</li>
	<li>Place all three K562 target cell controls in the incubator at 37 &#176;C for 30 min.</li>
	<li>After the 30-minute incubation has elapsed, centrifuge all three K562 target cell controls for 2 min at 163 x g.</li>
	<li>Carefully remove the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend each control with 20 &#181;L of fresh IMDM cell culture media, and leave in the 37 &#176;C incubator with 5% CO2 for at least 30 min for optimal DiO signal. 
	NOTE: Controls are now ready to be run through the imaging flow cytometer.</li>
</ol>
2. Cytotoxicity Assay Sample Preparation
<ol>
	<li>Prepare and label 1.5 mL tubes for each sample/participant accordingly.</li>
	<li>Pipette desired the ratio of NK cells and DiO-labeled K562 cells in each tube. 
	NOTE: For example, the desired ratio of K562 target cells and NK effector cells is 1:5.</li>
	<li>Centrifuge for 5 min at 135 x g.</li>
	<li>Carefully remove the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend NK-DiO-labeled K562 cell mixture in 500 &#181;L of NK cell media without interleukin-2 (IL-2) and 2-mercaptoethanol (2-ME) (incomplete NK cell culture media). 
	NOTE: The incomplete NK cell culture media is Minimum Essential Medium Eagle with sodium bicarbonate, without L-glutamine, ribonucleosides, and deoxyribonucleosides.</li>
	<li>Add 5 &#181;L of PI to each tube.</li>
	<li>Centrifuge for 2 min at 163 x g.</li>
	<li>Incubate cells at 37 &#176;C for 2 h.</li>
	<li>Following 2 h incubation, centrifuge for 2 min at 163 x g.</li>
	<li>Carefully remove the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend cells in 25 &#181;L incomplete NK cell culture media.</li>
</ol>
3. Preparation of Spontaneous (&#34;S&#34;) Sample
<ol>
	<li>Pipette 500 &#181;l of DiO-labeled K562 cells (concentration of 1 x 106 cells/mL) into a 1.5 mL tube.</li>
	<li>Add 10 &#181;L of PI to each tube.</li>
	<li>Centrifuge tube for 2 min at 163 x g.</li>
	<li>Incubate cells at 37 &#176;C for 2 h.</li>
	<li>Following 2 h incubation, centrifuge for 2 min at 163 x g.</li>
	<li>Carefully remove the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend cells in 25 &#181;l incomplete alpha-minimum Essential Medium (&#945;-MEM) cell culture media.</li>
</ol>
4. Data Acquisition with Imaging Flow Cytometer
<ol>
	<li>Press the green button inside the front door of the imaging flow cytometer to turn on the instrument.</li>
	<li>Turn on all computers associated with the imaging flow cytometer.</li>
	<li>Launch the imaging flow cytometer software.</li>
	<li>Click the &#34;Startup&#34; button to flush the system and prepare the sample line.</li>
	<li>Once the &#34;Startup&#34; is complete, close out the &#34;Calibrations&#34; window.</li>
	<li>Assign channels: on the top left-hand side, click on each channel in order to assign them.</li>
	<li>On the right-hand side, click on the scatter plot button to create 4 scatterplots: Raw Max Pixel _MC_6 vs Area_M06, Raw Max Pixel _MC_2 vs Area_M02, Raw Max Pixel _MC_5 vs Area_M05 and FieldArea_M01 vs AspectRatio_M01.</li>
	<li>Begin analyzing samples by first using the &#34;Double Positive&#34; control.
	<ol>
		<li>Click on &#34;Load.&#34;</li>
		<li>Place the 1.5 ml tube with the &#34;Double Positive&#34; sample from Steps 1.4 to 1.8 into the sample loader.</li>
		<li>Select the 40X objective under the &#34;Magnification&#34; tab.</li>
		<li>Turn on the 405 mW and 642 mW lasers.</li>
		<li>Turn on the &#34;Brightfield&#34; channel.</li>
		<li>Click on &#34;Select Intensity.&#34;</li>
		<li>Based on the &#34;Double Positive&#34; control sample, determine the desired intensity for the 405 mW laser so the detector is not overloaded. 
		Note: For example, the optimal intensity for this experiment was set at 11 mW.</li>
	</ol>
	</li>
	<li>After the desired set-up is achieved, acquire data.
	<ol>
		<li>Under the &#34;File Acquisition&#34; tab, enter a custom filename text. Select a folder for saving the data file(s).</li>
		<li>Enter the number of cells to acquire next to &#34;Collect&#34;. Typically this number varies between 1,000 to 10,000.</li>
		<li>Click on &#34;Acquire.&#34; 
		NOTE: Once the desired number of cells is acquired, the data file is automatically saved in the previously selected folder.</li>
	</ol>
	</li>
	<li>After acquisition finishes, load the next control sample &#8211; DiO only control.
	<ol>
		<li>Click on &#34;Load.&#34;</li>
		<li>Place the 1.5 mL tube with the &#34;DiO only&#34; sample into the sample loader.</li>
		<li>Leave the 40X objective under the &#34;Magnification&#34; tab selected.</li>
		<li>Leave the 405 mW laser turned on.</li>
		<li>Turn OFF the 642 mW laser and the &#34;Brightfield&#34; channel. 
		NOTE: Now that the desired set-up has been achieved, data can be acquired.</li>
		<li>Under the &#34;File Acquisition&#34; tab, enter a custom filename text and select a folder for saving the data file(s). Enter the number of cells to acquire next to &#34;Collect.&#34;. Typically this number is 1,000.</li>
		<li>Click on &#34;Acquire.&#34; 
		NOTE: Once the desired number of cells is acquired the data file is automatically saved in the previously selected folder.</li>
	</ol>
	</li>
	<li>Repeat step 4.10 for the &#34;PI only&#34; control sample. The experimental samples are now ready to be collected.</li>
	<li>Handle the remaining experimental samples, including the &#34;Spontaneous &#39;S&#39; Samples&#34; as follows:
	<ol>
		<li>Leave the 40X objective under the &#34;Magnification&#34; tab selected.</li>
		<li>Turn ON the 405 mW and 642 mW lasers.</li>
		<li>Turn ON the &#34;Brightfield&#34; channel.</li>
		<li>Click on &#34;Set Intensity.&#34;</li>
		<li>Under the &#34;File Acquisition&#34; tab, enter a custom filename text and select a folder for saving the data file(s). Enter a custom filename text.</li>
		<li>Enter the number of cells to acquire next to &#34;Collect.&#34;</li>
		<li>Click on the &#34;Acquire&#34; button. 
		NOTE: Once the desired number of cells is acquired the data file is automatically saved in the previously selected folder.</li>
	</ol>
	</li>
	<li>Repeat step 4.12 for all experimental samples.</li>
	<li>After all experimental data and files have been collected, click the &#34;Shutdown&#34; button to sterilize the system.</li>
</ol>
5. Imaging Flow Cytometer Sample Analysis
<ol>
	<li>Open the imaging flow cytometer analysis software application.</li>
	<li>Under &#34;File&#34;, open an experimental.RIF file.</li>
	<li>Build a new matrix using a single color.RIF files (DiO-only control and PI-only control, created during steps 4.10 and4.11) by selecting &#34;Create a New Matrix&#34; under the Compensation tab in the imaging flow cytometer software. 
	NOTE: The software will prompt for the selection of the single color files and merge them to create a matrix file (.ctm file extension), that is to be selected to apply channel compensation.</li>
	<li>Create dot plots by using the &#34;building blocks&#34; function of the software.
	<ol>
		<li>Create a dot-plot (BrightFieldGradient_RMS vs frequency) to gate the focused cells. Call the gate &#34;Focus&#34; (Figure 1A).</li>
		<li>Using the &#34;Focus&#34; gate, create a dot plot of Bright Field Area vs Bright Field Aspect Ratio to gate the singlet cells. Call the gate &#34;Single&#34; (Figure 1B).</li>
		<li>Using the &#34;Single&#34; gate, create a dot plot of Intensity_MC_Ch02 vs Intensity_MC_Ch5. Use this plot to identify and gate DiO-positive only cells (targets, alive) and PI-DiO double-positive cells (targets, dead) (Figure 1C). 
		NOTE: All the plots described in steps 5.4.1, 5.4.2, and 5.4.3 can be created by using the &#34;building blocks&#34; function of the software.</li>
	</ol>
	</li>
	<li>Click on the statistics function of the dot plot to access the cell numbers of each gate.</li>
	<li>Calculate the percentage of dead targets in the spontaneous sample and experimental samples using the following formula: 
	% dead targets in sample = (#dead targets x 100)/(#live targets + #dead targets)</li>
	<li>Calculate cytotoxicity using the following formula: 
	% cytotoxicity = [(Experimental dead-Spontaneous dead)/(100-Spontaneous dead)]x100</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>K-562 lymphoblasts</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Media</td>
			<td>ATCC</td>
			<td>30-2005</td>
			<td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential medium</td>
			<td>ATCC</td>
			<td>CRL-2407</td>
			<td>Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Propridium Iodide Staining Solution</td>
			<td>BD Pharmingen</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Vybranto DiO cell-labeling solution</td>
			<td>Vybranto</td>
			<td>V-22886</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageStream X Mark II Imaging Flow Cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood CD56 MicroBeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-875</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageStream X Mark II Imaging Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Speedbeads</td>
			<td>Amnis Corporation</td>
			<td>400030</td>
			<td></td>
		</tr>
		<tr>
			<td>INSPIRE Software</td>
			<td>EMD Millipore</td>
			<td>Version Mark II, September 2013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ideas Application Software</td>
			<td>EMD Millipore</td>
			<td></td>
			<td>Version 6.1, July 2014</td>
		</tr>
	</tbody>
</table>

natural killer cell-mediated cytotoxicity assay sample preparation for flow cytometric analysis

Source: McBride, J. E., et al. <a href="https://app.jove.com/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

<img alt="Figure 1" src="/files/ftp_upload/21846/21846_Figure1.jpg" /> 
Figure 1: Representative histograms, scatter plots, and images for cytotoxic activity analysis. (A) focus cell analysis. (B) single cell analysis. (C) target cell staining analysis. All determinations are made using the image attached to each event. This can be accessed in analysis software by simply clicking on the event on the graphs. (...

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

1.&#160;Preparation of Controls

	Transfer the following into separate and appropriately labeled 1.5 ml tubes:
	
		Add 500 &#181;L of fresh IMDM ...

Begin with a cell pellet containing natural killer or NK cells, and fluorescently stained target cells.
Resuspend the pellet in a suitable medium and incubate.
The NK cell interacts via surface adhesion proteins, forming an immunological synapse with the target cell.
This initiates NK-cell signaling, releasing cytotoxic granules, containing perforin and granzymes into the synapse.
Perforin, a pore-forming protein, creates channels in the target cell&#39;s membrane, allowing granzymes to enter the cytoplasm.
Granzyme, a serine protease, interacts with intracellular proteins, activating the apoptotic pathway in the target cell.
Now, add propidium iodide fluorescent dye.
The dye enters dead cells through their damaged membranes and binds to the DNA by intercalating between its base pairs.
Consequently, the dead cells express double fluorochrome while the live cells express single fluorochrome.
The sample is ready for flow cytometry to assess NK cell-induced cytotoxicity by quantifying dead and live target cell numbers.

natural-killer-cell-mediated-cytotoxicity-assay-sample-preparation

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

137 Görüntüleme. Kaynak: McBride, J. E., et al. İnsan Tam Kanında Doğal Öldürücü Hücre Litik Aktivitesinin Sitometrik Analizi. J. Vis. Uzm. (2017). Bu video, doğal öldürücü hücrelerin floresan lekeli hedef hücrelere karşı litik aktivitesini göstermektedir. Yöntem, ölü hücreleri boyayarak ve canlı ve ölü hedef hücreleri ölçmek için bir akış sitometresi kullanarak NK hücresinin neden olduğu sitotoksisite ölçümünü mümkün kılar. 

Video: Akış Sitometrik Analizi için Doğal Öldürücü Hücre Aracılı Sitotoksisite Testi Numune Hazırlama - Kavram

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Görüntüleme Sitometrisi ile İnsan Monosit türetilmiş dendritik hücrelerle Karakterizasyonu: İki Monosit İzolasyon Protokoller Arasında Bir Karşılaştırma

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

JoVE Journal

İmmünoloji ve Enfeksiyon

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Akış NK Hücre Fonksiyonları İzleme Testi Sitometri tabanlı

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

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Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis