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 manufacturer&#39;s instructions. Keep blood collection tube at room temperature on a bench-top rocker until separation.</li>
</ol>
2. Preparation of DiO-labeled Target Cells
<ol>
	<li>Grow K562 cells in Complete Iscove&#39;s Modified Dulbecco&#39;s Media (IMDM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin for several weeks prior to the assay to ensure the health of the cells. Adjust the concentration of the cells to 3 x 105 cells/mL daily through the completion of a cell count and subsequent passage of cells. Perform a complete media change every 2 to 3 days.</li>
	<li>On the day of the assay, perform a cell count using a 1:1 hemocytometer.
	<ol>
		<li>Remove 10 &#181;L from the K562 flask and place into 1.5 mL tube.</li>
		<li>Add 10 &#181;L of trypan blue dye into the same 1.5 ml tube for a dilution factor of 1:1.</li>
		<li>Gently flick tube to mix K562 cells and trypan blue dye.</li>
		<li>Allow K562 cell-trypan blue dye to incubate for 1 min at room temperature.</li>
		<li>Remove 15 &#181;L of stained K562 cells from tube.</li>
		<li>Pipette onto hemocytometer for cell count.</li>
		<li>Count cells in the four corner squares as well as the middle square for a total of five squares.</li>
		<li>Calculate cell count by using the equation: 
		Total cells/mL = Total cells counted X (dilution factor/Number of squares) X 10,000/mL</li>
	</ol>
	</li>
	<li>Resuspend K562 target cells at a final density of 1 x 106 cells/ml in serum free IMDM culture medium.</li>
	<li>For unstained K562 target cells, add 10 mL of K562 target cells to one 15 mL tube at a density of 1 x 106 cells/mL for a total of 10 x 106 K562 cells. Place into a 37 &#176;C incubator with 5% CO2 until ready for use.</li>
	<li>For DiO stained K562 target cells, add 10 mL of K562 targets cells to one 15 mL tube at a density of 1 x 106 cells/mL for a total of 10 x 106 K562 cells.
	<ol>
		<li>Add 1 &#181;L of DiO cell-labeling solution per mL of cell suspension in 15 ml tube designated for DiO staining and gently vortex. For example, add 10 &#181;L of DiO cell-labeling solution to 10 x 106 K562 cells/ml at a volume of 1 x 106 cells/mL.</li>
		<li>Incubate K562-DiO solution for 20 min at 37 &#176;C with 5% CO2 in a 15 ml tube.</li>
	</ol>
	</li>
	<li>Following incubation, add 3 ml of room temperature phosphate-buffered saline (PBS) to K562-DiO solution containing 15 ml tube.</li>
	<li>Centrifuge for 10 min at 135 x g at room temperature.</li>
	<li>Carefully remove supernatant without disturbing the cell pellet with a 1,000 &#181;l volume-adjustable pipette.</li>
	<li>Add 10 mL of fresh IMDM to cell pellet-containing 15 mL tube.</li>
	<li>Gently vortex tube to resuspend the cells.</li>
	<li>Repeat steps 2.7 to 2.10 two more times.</li>
	<li>Store cells in a 37 &#176;C incubator with 5% CO2 until ready for use. 
	NOTE: Cells can be stored in the incubator for up to 24 h but it is preferable to use them on the same day.</li>
</ol>
3. 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 tubes and wipe 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 min incubation has elapsed, centrifuge all three K562 target cell controls for 2 min at 163 x g.</li>
	<li>Carefully remove 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>
4. NK Cell Automated Separation
<ol>
	<li>Turn on cell separator and allow the start-up cycle to finish.</li>
	<li>Ensure that all fluid bottles illuminations are green and that the waste bottle is empty.</li>
	<li>Obtain a room temperature 15 mL tube rack. 
	NOTE: Selection is based on sample volumes. For example, for a volume less than 3 ml use a 15 ml tube rack and for a volume more than 3 mL use a 50 mL tube rack.</li>
	<li>Label sample tubes accordingly (Repeat per sample/participant): Participant 1 Whole Blood Sample; Participant 1 Negative fraction; Participant 1 Positive fraction.</li>
	<li>Gently pipette 1.5 mL of whole blood from Step 1.2 into &#34;Whole Blood Sample&#34; 15 mL tube.</li>
	<li>Place appropriately labeled 15 mL &#34;Whole Blood Sample&#34; tube from Step 4.5 and labeled 15 mL &#34;Negative Fraction&#34; and &#34;Positive Fraction&#34; tubes from Step 4.4 in the tube rack. Use thefollowing sample rack set-up: Row (R1) A: Whole Blood Sample, Row (R2) B: Negative, unlabeled fraction, Row (R4) C: Positive, magnetically labeled fraction.</li>
	<li>Insert the separator rack onto the MiniSampler for autolabeling.</li>
	<li>Select &#34;Reagent&#34; on the menu and highlight the position where the vial will be placed in the separator rack.</li>
	<li>Select &#34;Read Reagent&#34; to activate the 2D code reader.</li>
	<li>Place the appropriate reagent vial in front of the 2D code reader between 0.5-2.5 cm from the code reader cover. 
	NOTE: For example, the required reagent for NK cell separation is CD56.</li>
	<li>Hold reagent vial at an angle in front of 2D code reader for optimal reading.</li>
	<li>Place the reagent vial into the proper separator rack position.</li>
	<li>Highlight the desired positions under the Separation tab on the screen.</li>
	<li>From the Labeling submenu, assign an autolabeling program.</li>
	<li>Assign the Cell Separation Reagent CD56 MicroBeads to rack positions 1, 2, 3, and 4.</li>
	<li>Select the &#34;posselwb&#34; separation program.</li>
	<li>Select the &#34;rinse&#34; wash program.</li>
	<li>Insert a sample volume of 1,500 &#181;L in the &#34;Volume&#34; submenu using the numeric keypad.</li>
	<li>Select the &#34;Enter&#34; option on the keypad.</li>
	<li>Select &#34;Run&#34; to start the cell separation.</li>
	<li>Select &#34;OK&#34; to confirm that enough buffer is available for processing all samples.</li>
</ol>
5. NK Cell Count Following Cell Separation
<ol>
	<li>Immediately following cell separation with the cell separator, retrieve the positive fraction. Leave at room temperature. This fraction contains the desired pure NK cell population.</li>
	<li>For each individual sample, perform a cell count using a hemocytometer as per Step 2.2.
	<ol>
		<li>After calculations, record the cell count.</li>
	</ol>
	</li>
</ol>
6. Cytotoxicity Assay Sample Preparation
<ol>
	<li>Prepare and label 1.5 mL tubes for each sample/participant accordingly.</li>
	<li>Pipette desired ratio of NK cells and DiO-labled K562 cells into 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 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 supernatant without disturbing the cell pellet.</li>
	<li>Resuspend cells in 25 &#181;L incomplete NK cell culture media.</li>
</ol>
7. 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 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 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>
8. 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 3.4 to 3.9 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 in 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 in 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 8.10 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 in a custom filename text and select a folder for saving the data file(s). Enter in 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 8.12 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>
9. 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 the single color .RIF files (DiO-only control and PI-only control, created during steps 8.10 and 8.11) 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 9.4.1, 9.4.2, and 9.4.3 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 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>
<img alt="Figure 1" src="/files/ftp_upload/54779/54779fig1.jpg" /> 
Figure 1: Representative histograms,scatter plots and images for cytotoxic activity analysis. (A)&#160;focus cell analysis. (B)&#160;single cell analysis.&#160;(C)&#160;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. (D)&#160;representative image of a doublet event, showing an apoptotic NK cell and a live K562 target. Ch01, Brightfield. Ch02, DiO. Ch05, PI. <a href="http://cloudfront.jove.com/files/ftp_upload/54779/54779fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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<li>Nieman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marathon+training+and+immune+function%5BTitle%5D)+AND+%22Sports+Med%22%5BJournal%5D)">Marathon training and immune function.</a> Sports Med. 37 (4-5), 412-415 (2007).</li>
<li>Simpson, R. J., Kunz, H., Agha, N., Graff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise+and+the+Regulation+of+Immune+Functions%5BTitle%5D)+AND+%22Prog+Mol+Biol+Transl+Sci%22%5BJournal%5D)">Exercise and the Regulation of Immune Functions.</a> Prog Mol Biol Transl Sci. 135, 355-380 (2015).</li>
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</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>K-562 lymphoblasts</td><td>ATCC</td><td>CCL-243</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;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>Trypan Blue Solution 0.4%</td><td>Amresco</td><td>K940-100ML</td><td>Tissue culture grade</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>autoMACS Pro Separator</td><td>Miltenyi Biotec</td><td>130-092-545</td><td></td></tr><tr><td>autoMACS Running Buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>autoMACS Washing Buffer</td><td>Miltenyi Biotec</td><td>130-092-987</td><td></td></tr><tr><td>autoMACS Columns</td><td>Miltenyi Biotec</td><td>130-021-101</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>0.4-0.7% Hypochlorite (Sterilizer)</td><td>VWR</td><td>JT9416-1</td><td></td></tr><tr><td>Coulter Clenz</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>70% Isopropanol (Debubbler)</td><td>EMD Millipore</td><td>1.3704</td><td></td></tr><tr><td>D-PBS (Sheath fluid)</td><td>EMD Millipore</td><td>BSS-1006-B (1X)</td><td>No calcium or magnesium</td></tr><tr><td>INSPIRE Software</td><td>EMD Millipore</td><td></td><td>Version Mark II, September 2013</td></tr><tr><td>Ideas Application Software</td><td>EMD Millipore</td><td></td><td>Version 6.1, July 2014</td></tr></tbody></table>

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...

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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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Research

JoVE Journal

Immunology and Infection

12.4K Views.  North Carolina A&T State University, North Carolina Research Campus. 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.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

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.

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

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Medicine

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.

1.&#160;Preparation of Controls

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

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

Source: Baik, C. H., et al. <a href="https://app.jove.com/v/58961">A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function.</a> J. Vis. Exp. (2019)
This video demonstrates performing a lactate dehydrogenase release assay to determine the cytotoxicity of natural killer (NK) cells against the target cancer cells. The assay quantifies the release of the cytoplasmic lactate dehydrogenase enzyme from cancer cells by measuring the absorbance of the red-colored product formed.

1. Cytotoxicity Assay: Assay Protocol

	Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows ...