Name: flow cytometric analysis of natural killer cell lytic activity in human whole blood
Uploaded: 2017-03-17T12:30:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood at JoVE.com

This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2100-68003-30395 from the USDA National Institute of Food and Agriculture.

NOTE: All blood collection procedures were conducted in accordance with the guidelines set forth by the Appalachian State University (ASU) Institutional Review Board (IRB).
1. Whole Blood Collection
<ol>
	<li>Have a certified phlebotomist draw blood according to World Health Organization&#39;s guidelines.</li>
	<li>Draw blood into one 4 ml blood collection tube containing Di-Potassium Ethylenediaminetetraacetic acid (K2EDTA). Invert blood collection tube according to the manufactu...

<ol>
	<li>Cerwenka, A., Lanier, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells,+viruses+and+cancer.">Natural killer cells, viruses and cancer.</a> Nat Rev Immunol. 1 (1), 41-49 (2001).</li><li>Nieman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise,+infection,+and+immunity.">Exercise, infection, and immunity.</a> Int J Sports Med. 15, 131-141 (1994).</li><li>Romeo, J., Warnberg, J., Pozo, T., Marcos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity,+immunity+and+infection.">Physical activity, immunity and infection.</a> Proc Nutr Soc. 69 (3), 390-399 (2010).</li><li>Nieman, D. 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The method described in this study directly measures the specific activity of an individual&#39;s NK cells in response to stimuli (in this particular case, prolonged exercise). Typically, NK cells are isolated from one&#39;s blood using density gradients or cell sorting by using a combination of markers. While these methods are widely used, they have many drawbacks: they are time consuming, involve multiple manipulations, and are heavily user dependent. As a result, undue stress is placed on the isolated NK cells, which ...

Natural killer cells are an essential element of the innate immune system. While they are very regulated, they have the capability to recognize and eliminate abnormal cells through cell-to-cell contact and without prior activation1. As such, they form a quick line of defense against infections. Exercise, especially intense, has been shown to transiently depress the immune system2,3,4,5. NK cells are particularly prone to this effect4,6,7, effectively creating a window of enhanced sensitivity to disease. Hence, the study of interventions before, during or after intense exercise with the goal of reducing its impact on NK cell function is of particular interest for the well-being of athletes post-competitions.
However, the study of such interventions is complicated by numerous factors: 1)&#160;NK cells are sparse8, at about 1% of the white blood cell compartment; 2)&#160;NK cells are very sensitive to stress and rely on constant exposure to physiological conditions to remain viable and stable during experimentation; and 3)&#160;standard assays to determine NK cell cytotoxicity, such as Ficoll gradients9 and release assays10, are unreliable and inconsistent. The inherent variability of human samples only compound these issues. Fresh human samples collected during interventions are fairly regulated and difficult to procure, at least when compared to animal samples or immortalized cell lines. This reduces opportunities to repeat experiments or add participants to the study cohort to reach significant statistical thresholds. Collectively, these issues support the need for a streamlined protocol that allows for both high-throughput and a high-reliability analysis of NK cell lytic activity in human samples.
We established a workflow that shortens the time necessary to identify, isolate and test NK cells from human whole blood while minimizing exposure to extraneous factors. The method optimizes the use of two instruments, a magnetic-based cell sorter and an imaging flow cytometer, and an assay-specific, optimized T:E ratio to allow the detection of decreases or increases of NK cell cytotoxicity.

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			<td height="21" style="height:21px;width:371px;">K-562 lymphoblasts</td>
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			<td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td>
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			<td height="21" style="height:21px;width:371px;">Trypan Blue Solution 0.4%</td>
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			<td>Tissue culture grade</td>
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			<td height="21" style="height:21px;width:371px;">Propridium Iodide Staining Solution</td>
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			<td height="21" style="height:21px;width:371px;">Vybranto DiO cell-labeling solution</td>
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			<td></td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Pro Separator</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-545</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Running Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Washing Buffer</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Columns</td>
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			<td height="21" style="height:21px;">Whole Blood CD56 MicroBeads, human</td>
			<td style="width:183px;">Miltenyi Biotec</td>
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			<td height="21" style="height:21px;">ImageStream X Mark II Imaging Flow Cytometer</td>
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			<td height="21" style="height:21px;">Speedbeads</td>
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			<td style="width:136px;">400030</td>
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			<td height="21" style="height:21px;">0.4-0.7% Hypochlorite (Sterilizer)</td>
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			<td height="21" style="height:21px;">Coulter Clenz</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td>8546929</td>
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			<td height="21" style="height:21px;">70% Isopropanol (Debubbler)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">1.3704</td>
			<td></td>
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			<td height="21" style="height:21px;">D-PBS (Sheath fluid)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">BSS-1006-B (1X)</td>
			<td>No calcium or magnesium</td>
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			<td height="21" style="height:21px;">INSPIRE Software</td>
			<td style="width:183px;">EMD Millipore</td>
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			<td>Version Mark II, September 2013</td>
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			<td height="21" style="height:21px;">Ideas Application Software</td>
			<td style="width:183px;">EMD Millipore</td>
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			<td>Version 6.1, July 2014</td>
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flow cytometric analysis of natural killer cell lytic activity in human whole blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

Determination of NK cell count 
The effect of heavy running on NK cell count in whole blood was measured, using the exercise protocol described in Figure 2. Blood samples were drawn before exercise, immediately after exercise, 1.5 h after exercise, and finally 24 and 48 h after the initial blood draw. The concentration of NK cells per milliliter of whole blood was measured for each runner (Figure 3A) and on average (Figure 3B) for ea...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood at JoVE.com

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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