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>

<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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marathon+training+and+immune+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+and+the+Regulation+of+Immune+Functions.">Exercise and the Regulation of Immune Functions.</a> Prog Mol Biol Transl Sci. 135, 355-380 (2015).</li><li>Walsh, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position+statement.+Part+one:+Immune+function+and+exercise.">Position statement. Part one: Immune function and exercise.</a> Exerc Immunol Rev. 17, 6-63 (2011).</li><li>Timmons, B. W., Cieslak, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+natural+killer+cell+subsets+and+acute+exercise:+a+brief+review.">Human natural killer cell subsets and acute exercise: a brief review.</a> Exerc Immunol Rev. 14, 8-23 (2008).</li><li>Westermann, J., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+lymphocyte+subsets+and+natural+killer+cells+in+the+human+body.">Distribution of lymphocyte subsets and natural killer cells in the human body.</a> Clin Investig. 70 (7), 539-544 (1992).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+leucocytes+from+human+blood.+Further+observations.+Methylcellulose,+dextran,+and+ficoll+as+erythrocyteaggregating+agents.">Isolation of leucocytes from human blood. 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A., Travers, P., Walport, M., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. 5th edn. , (2001).</li></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 border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">K-562 lymphoblasts</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CCL-243</td>
			<td style="width:585px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Media</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">30-2005</td>
			<td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Alpha Minimum Essential medium</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CRL-2407</td>
			<td style="width:585px;">Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trypan Blue Solution 0.4%</td>
			<td style="width:183px;">Amresco</td>
			<td style="width:136px;">K940-100ML</td>
			<td>Tissue culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Propridium Iodide Staining Solution</td>
			<td style="width:183px;">BD Pharmingen</td>
			<td style="width:136px;">51-66211E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Vybranto DiO cell-labeling solution</td>
			<td style="width:183px;">Vybranto</td>
			<td style="width:136px;">V-22886</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Running Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Washing Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Columns</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Blood CD56 MicroBeads, human</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-090-875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageStream X Mark II Imaging Flow Cytometer</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Speedbeads</td>
			<td style="width:183px;">Amnis Corporation</td>
			<td style="width:136px;">400030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4-0.7% Hypochlorite (Sterilizer)</td>
			<td style="width:183px;">VWR</td>
			<td>JT9416-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coulter Clenz</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td>8546929</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INSPIRE Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td>Version Mark II, September 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ideas Application Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></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...

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 ...

flow-cytometric-analysis-natural-killer-cell-lytic-activity-human

Research

JoVE Journal

Immunology and Infection

12.3K 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

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...