eoe sub articles

EoE Sub Articles

1.&#160;Preparation of Controls
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	<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...

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.