Name: a flow cytometry-based cytotoxicity assay for the assessment of human nk cell activity
Uploaded: 2017-08-09T14:00:00.000+00:00
Duration: 6 min 8 s
Description: Watch this Scientific Journal Video about A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity at JoVE.com

Wir möchten Jill Narciso, UCLA Immungenetik Center, danke für ihre Unterstützung bei der Vorbereitung der Handschrift.

Samples were collected according to the ethical guidelines established by the UCLA Human Research Protection Program and IRB approved.
1. Preparation of reagents
NOTE: Unless otherwise stated, all reagents should be allowed to equilibrate at room temperature prior to use. All reagents must remain sterile.
<ol>
	<li>Prepare a 2x working solution of Tween-20 (i.e., 0.2%) by adding 10&#160;&#181;L of Tween-20 solution to 5 mL of phosphate-buffered saline (PBS) without calcium and magnesium with a p20 pipette.
	<ol>
		<li>Given the high viscosity of Tween-20, take the following steps to ensure accuracy: collect slowly to avoid bubble formation, do not submerge the entire tip to avoid carry-over, and pipette up and down several times in PBS to wash out all Tween-20 from the tip.</li>
	</ol>
	</li>
	<li>Add 2 &#181;L of IL-2 stock solution (2.1E6 IU/mL) to 198 &#181;L of complete media (i.e., 1:100 dilution), which is RPMI with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS), then proceed to an additional 1:100 dilution by adding 2 &#181;L of IL-2 intermediate solution to 198 &#181;L of complete media to prepare a 7x working solution (210 IU/mL).</li>
	<li>Add 2 &#181;L of CFSE stock solution (5 mM) to 500 &#181;L of plain media (RPMI). Vortex, spin down to remove drops from the cap, then proceed with an additional 1:20 dilution by adding 50 &#181;L of the CFSE intermediate solution to 950 &#181;L of plain RPMI to obtain a 2x working solution of 1 &#181;M. Vortex well and keep protected from light.</li>
</ol>
2. Isolation of PBMCs as effector cells
NOTE: This assay has been validated for effective use with total PBMCs from healthy controls. However, it is recommended that NK cell content be verified with each PBMC preparation (Figure 1). Also, the volume of whole blood for collection is based on the frequency of NK cells in peripheral whole blood and this may vary from person to person, particularly between healthy donors and patients.
<ol>
	<li>Collect a minimum of 4 mL of human whole blood in sodium or lithium heparinized blood collection tubes. Store blood samples at room temperature upon collection and use within 30 h of collection.</li>
	<li>Dilute whole blood with 4 mL of PBS (i.e. equal volume of PBS to whole blood).</li>
	<li>Add 4 mL of density gradient solution (see table of materials) to a 15-mL conical tube. Carefully overlay diluted whole blood over the density gradient solution by tilting the tube.</li>
	<li>Centrifuge at 650 x g for 24 min with the brake off.</li>
	<li>Carefully collect the layer of mononuclear cells, which is the thin white layer below the top layer of plasma and platelets, into a new 15-mL conical tube. Bring the volume to 15 mL with PBS.</li>
	<li>Centrifuge at 450 x g for 10 min with the brake on. Aspirate the supernatant and resuspend cells in 0.5 mL of complete media.</li>
	<li>Use an automated cell counter or hemocytometer to count the cells, and adjust the PBMC&#160;concentration to 5x106 cells/mL with complete media.</li>
	<li>Check the NK cell content by flow cytometry (see step 4).</li>
	<li>Start the assay within 30 min. Keep cells at room temperature for up to 10 min, or place in a humidified 5% CO2 incubator at 37 &#176;C until ready for use.</li>
</ol>
3. Isolation of NK cells as effector cells
NOTE: This portion of the protocol is an alternative to using total PBMCs as effector cells. The typical yield from 4 mL of whole blood from a healthy individual is approximately 4x105 NK cells, or approximately 5-15% of PBMCs, though this frequency varies between donors 21. It is recommended that a purity check be performed after the isolation.
<ol>
	<li>Collect a minimum of 4 mL of human whole blood in sodium or lithium heparinized blood collection tubes. Take 50 &#181;L of whole blood to stain as a pre-enrichment sample.</li>
	<li>Add the enrichment cocktail to blood samples at 50 &#181;L/mL (see table of materials). Mix well and incubate for 10 min at room temperature.</li>
	<li>Add an equal volume of PBS + 2% FBS to dilute the sample and gently mix well.</li>
	<li>Add 4 mL of density gradient solution to a 15-mL conical tube. Carefully overlay the diluted sample on top of the density gradient medium.</li>
	<li>Centrifuge at 1,200 x g for 10 min with the brake off.</li>
	<li>Carefully collect the layer of enriched cells into a new 15-mL conical tube. Wash the cells by bringing the volume up to 50 mL with PBS + 2% FBS.</li>
	<li>Centrifuge at 300 x g for 10 min. Discard the supernatant and repeat the wash.</li>
	<li>Aspirate the supernatant and resuspend cells in 0.5 mL of complete media.</li>
	<li>Using an automated cell counter or hemocytometer, count the cells and adjust the concentration to 5x105 cells/mL. Take 50 &#181;L of purified cell suspension to stain as a post-enrichment sample.</li>
	<li>Keep cells at room temperature for up to 10 min, or place in a humidified 5% CO2 incubator at 37 &#176;C until ready for use.</li>
</ol>
4. Immunophenotyping for assessment of NK cell content
<ol>
	<li>Using 50 &#181;L total isolated PBMCs, pre-enrichment whole blood, or purified NK cells, stain with anti-CD56 fluorochrome-conjugated antibody as an NK cell marker. Markers for other cell types and lineages (i.e. CD3+ T cells, CD19+ B cells) may also be included to assess the origin of contamination, if any.</li>
	<li>Incubate for 20 min at 4 &#176;C, and wash by adding 2 mL of PBS + 2% FBS.</li>
	<li>Centrifuge at 450 x g for 5 min.</li>
	<li>For PBMCs or NK cells, aspirate supernatant and resuspend cells in 100 &#181;L of PBS + 2% FBS.</li>
	<li>For whole blood samples, perform red blood cell (RBC) lysis:
	<ol>
		<li>After centrifugation, aspirate the supernatant and resuspend in 2 mL red blood cell lysis buffer (31.75 g of ammonium chloride + 4.16 g of Tris base + 6.65 g of EDTA, dissolved in 5000 mL of deionized water).</li>
		<li>Incubate for 10 min at room temperature.</li>
		<li>Repeat wash twice, aspirate the supernatant, and resuspend lysed sample in 100 &#181;L of PBS + 2% FBS. 
		NOTE: RBC lysis may also be performed prior to staining.</li>
		<li>Proceed with flow cytometric analysis.</li>
	</ol>
	</li>
</ol>
5. Thawing, culturing, and harvesting of K562 target cells
<ol>
	<li>Aliquot 10 mL of complete media into a 15-mL conical tube and allow to warm in a 37 &#176;C water bath. For the thawing and culturing of K562 cells, use only media pre-warmed to 37 &#176;C for all steps.</li>
	<li>Place one vial of frozen K562 cells in the 37 &#176;C water bath until most of it is thawed.</li>
	<li>Transfer the semi-thawed K562 cells to the complete media, pre-warmed to 37 &#176;C, in the 15-mL conical tube. Wash the inside of the cryogenic vial with some pre-warmed media, and add the wash to the 15-mL tube.</li>
	<li>Centrifuge at 140 x g for 7 min with the brake on. Remove the supernatant and resuspend the pellet in 1 mL of complete media.</li>
	<li>Aliquot an appropriate amount of cell suspension to count cells using an automated cell counter or hemocytometer.</li>
	<li>Adjust the concentration to 1x105-2x105 cells/mL&#160;and culture in a humidified 5% CO2 incubator at 37 &#176;C for at least 2 days before use. Maintain the culture at this density range for no longer than 3 months, then thaw a new aliquot.&#160;Never exceed a cell concentration of 1x106 cells/mL in culture.</li>
	<li>For use in the assay, mix the K562 cell culture well with a 10-mL serological pipette and remove an aliquot to count and assess cell viability using an automated cell counter or hemocytometer and Trypan Blue staining.
	<ol>
		<li>If the viability is less than 85%, proceed with the following dead cell removal steps.
		<ol>
			<li>Add 15 mL of density gradient solution to 50-mL conical tubes, as needed. Collect all cells from culture and slowly overlay the cell suspension over the density gradient solution by tilting the tube.</li>
			<li>Centrifuge at 650 x gfor 24 min with brake off. Carefully collect the layer of live cells into a new 15-mL conical tube. Bring the volume to 50 mL with PBS.</li>
			<li>Centrifuge at 450 x g for 10 min with the brake on. Remove the supernatant and resuspend the pellet in 1 mL of complete media. Count the cells and check cell viability (as in 5.7).</li>
			<li>Culture in a humidified 5% CO2 incubator at 37 &#176;C for at least 2 days before use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect 5x105 K562 target cells and centrifuge at 140 x g for 7 min. Aspirate the supernatant and resuspend the cell pellet in 500 &#181;L in media with 1% FBS.</li>
</ol>
6. Labeling of K562 target cells
NOTE: Make sure the K562 cells are well resuspended before adding the CFSE working solution.
<ol>
	<li>Pipette 500 &#181;L of the CFSE working solution to the 500 &#181;L of K562 cells for a final concentration of 0.5 &#181;M. Using the same 1000-1250 &#181;L tip, mix immediately by gently pipetting up and down 3-5 times.</li>
	<li>Incubate in the 37 &#176;C water bath or in a humidified 5% CO2 incubator at 37 &#176;C for 10 min in the dark. 
	NOTE: Since the temperature equilibration lag time is longer in the incubator compared to the water bath, incubation time might need to be adjusted especially for volumes &#62; 1 mL.</li>
	<li>Quench the labeling reaction with 10 mL of complete media for 10 min in the dark at room temperature.</li>
	<li>Centrifuge the labeled K562 cells at 140 x g for 7 min. Remove the supernatant and resuspend in 1 mL of complete media.</li>
	<li>Count the cells using an automated cell counter or hemocytometer and bring the concentration to 1x105 cells/mL in complete media.</li>
	<li>Place cells in a humidified 5% CO2 incubator at 37 &#176;C until ready for use. Cells can be kept at room temperature for up to 10 min, or in in a humidified CO2 incubator at 37 &#176;C until ready for use. Do not refrigerate.</li>
</ol>
7. Plating
NOTE: If NK cells are limiting, the concentration of both K562 and NK cells can be halved, so that half of the cells are plated in the assay. The assay yields comparable results with half the amount of cells (as long as the ratios are kept consistent).
<ol>
	<li>If using total PBMCs, label a 96-well, U-bottom, tissue culture-treated plate for the following conditions per well: (1) Effector (E) : Target (T) = 50 : 1 + IL-2 - positive control for NK cell cytotoxicity; (2) E : T = 50 : 1; (3) E : T = 25 : 1; (4) E : T = 12.5 : 1; (5) E : T = 6.25 : 1; (6) T - target cells only - negative control for K562 death; (7) E - effector cells only; (8) T + Tween - Positive control for K562 death</li>
	<li>If using purified NK cells, label a 96-well, U-bottom, tissue culture treated plate for the following conditions per well: (1) Effector (E) : Target (T) = 5: 1&#160;+ IL-2 - positive control for NK cell cytotoxicity; (2) E : T = 5 : 1; (3) E : T = 2.5 : 1; (4) E : T = 1.25 : 1; (5) E : T = 0.625: 1; (6) T - target cells only - negative control for K562 death; (7) E - effector cells only; (8) T + Tween - positive control for K562 death</li>
	<li>Add 100 &#181;L of complete media to conditions 3, 4, 5, 6, 7. For this and all subsequent steps, (recommended) use a multichannel pipette.</li>
	<li>Add 100 &#181;L of effector cells (PBMCs at 5x106 cells/mL or purified NK cells at 5x105 cells/mL) to conditions 1, 2, 3. Add effector cells for conditions 4-7 by serial dilution. Do not change tips from condition to condition.
	<ol>
		<li>Mix condition 3, take 100 &#181;L, and transfer to condition 4.</li>
		<li>Mix condition 4, take 100 &#181;L, and transfer to condition 5.</li>
		<li>Mix condition 5, take 100 &#181;L, and transfer to condition 7; Condition 6 does not have effector cells.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of prepared 2x 0.2% Tween working solution to condition 8 (final concentration: 0.1% Tween-20); condition 8 does not have effector cells.</li>
	<li>Add 30 &#181;L of IL-2 working solution to condition 1. IL-2 should be added to the effector cell suspension prior to the addition of target cell suspension (final concentration: 27 IU/mL).</li>
	<li>Add 100 &#181;L of target cells (labeled K562 cells at 1x105 cells/mL) to conditions 1, 2, 3, 4, 5, 6, 8. Condition 7 does not have target cells.</li>
	<li>Mix all wells with a multichannel pipette and centrifuge the plate at 120 x g for 2 min.</li>
	<li>Incubate in a humidified CO2 incubator at 37 &#176;C for 4 h. At the end of the incubation, place the plate on ice and proceed to viability staining within 30 min.</li>
</ol>
8. Staining for viability and acquisition
<ol>
	<li>Prepare dead cell stain (see table of materials) by adding 3 &#181;L of the 5 &#181;M stock solution to 600 &#181;L of PBS.</li>
	<li>Add 50 &#181;L of diluted dead cell stain to each well for a final volume of 250 &#181;L and a final concentration of 0.005 &#181;M. Mix well with a multichannel pipette.</li>
	<li>Proceed with flow cytometric analysis. Using a flow cytometer, collect at least 1,000 events in the target cell gate (Figure 2) for statistically meaningful results.</li>
</ol>

<ol>
	<li>Iannello, A., Debbeche, O., Samarani, S., Ahmad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+NK+cell+responses+in+HIV+infection:+I.+NK+cell+receptor+genes+as+determinants+of+HIV+resistance+and+progression+to+AIDS.">Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS.</a> J Leukoc Biol. 84 (1), 1-26 (2008).</li><li>Caligiuri, M. A. <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+cells.">Human natural killer cells.</a> Blood. 112 (3), 461-469 (2008).</li><li>Topham, N. J., Hewitt, E. W. <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+cell+cytotoxicity:+how+do+they+pull+the+trigger?.">Natural killer cell cytotoxicity: how do they pull the trigger?.</a> Immunology. 128 (1), 7-15 (2009).</li><li>Warren, H. S., Smyth, 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=NK+cells+and+apoptosis.">NK cells and apoptosis.</a> Immunol Cell Biol. 77 (1), 64-75 (1999).</li><li>Tognarelli, S., Jacobs, B., Staiger, N., Ullrich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry-based+Assay+for+the+Monitoring+of+NK+Cell+Functions.">Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.</a> J Vis Exp. (116), (2016).</li><li>Somanchi, S. S., McCulley, K. J., Somanchi, A., Chan, L. L., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+for+Assessment+of+Natural+Killer+Cell+Cytotoxicity+Using+Image+Cytometry.">A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.</a> PLoS One. 10 (10), e0141074 (2015).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. 294 (1-2), 15-22 (2004).</li><li>Atkinson, E. A., Gerrard, J. M., Hildes, G. E., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+mechanism+of+natural+killer+(NK)+degranulation+and+cytotoxicity.">Studies of the mechanism of natural killer (NK) degranulation and cytotoxicity.</a> J Leukoc Biol. 47 (1), 39-48 (1990).</li><li>Kim, G. G., Donnenberg, V. S., Donnenberg, A. D., Gooding, W., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells:+comparisons+to+a+4+h+51Cr-release+assay.">A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.</a> J Immunol Methods. 325 (1-2), 51-66 (2007).</li><li>Kane, K. L., Ashton, F. A., Schmitz, J. L., Folds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+natural+killer+cell+function+by+flow+cytometry.">Determination of natural killer cell function by flow cytometry.</a> Clin Diagn Lab Immunol. 3 (3), 295-300 (1996).</li><li>Jang, Y. Y., 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=An+improved+flow+cytometry-based+natural+killer+cytotoxicity+assay+involving+calcein+AM+staining+of+effector+cells.">An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.</a> Ann Clin Lab Sci. 42 (1), 42-49 (2012).</li><li>Korzeniewski, C., Callewaert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-release+assay+for+natural+cytotoxicity.">An enzyme-release assay for natural cytotoxicity.</a> J Immunol Methods. 64 (3), 313-320 (1983).</li><li>Karimi, M. A., 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=Measuring+cytotoxicity+by+bioluminescence+imaging+outperforms+the+standard+chromium-51+release+assay.">Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.</a> PLoS One. 9 (2), e89357 (2014).</li><li>Oppenheim, D. E., 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=Glyco-engineered+anti-EGFR+mAb+elicits+ADCC+by+NK+cells+from+colorectal+cancer+patients+irrespective+of+chemotherapy.">Glyco-engineered anti-EGFR mAb elicits ADCC by NK cells from colorectal cancer patients irrespective of chemotherapy.</a> Br J Cancer. 110 (5), 1221-1227 (2014).</li><li>West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., Herberman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+cytotoxic+reactivity+of+human+lymphocytes+against+a+myeloid+cell+line:+characterization+of+effector+cells.">Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.</a> J Immunol. 118 (1), 355-361 (1977).</li><li>Jedema, I., van der Werff, N. M., Barge, R. M., Willemze, R., Falkenburg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+CFSE-based+assay+to+determine+susceptibility+to+lysis+by+cytotoxic+T+cells+of+leukemic+precursor+cells+within+a+heterogeneous+target+cell+population.">New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population.</a> Blood. 103 (7), 2677-2682 (2004).</li><li>Lecoeur, H., Fevrier, M., Garcia, S., Riviere, Y., Gougeon, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometric+assay+for+quantitation+and+multiparametric+characterization+of+cell-mediated+cytotoxicity.">A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.</a> J Immunol Methods. 253 (1-2), 177-187 (2001).</li><li>Carotta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NK+Cells+for+Anticancer+Immunotherapy:+Clinical+and+Preclinical+Approaches.">Targeting NK Cells for Anticancer Immunotherapy: Clinical and Preclinical Approaches.</a> Front Immunol. 7, 152 (2016).</li><li>Mandal, A., Viswanathan, C. <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:+In+health+and+disease.">Natural killer cells: In health and disease.</a> Hematol Oncol Stem Cell Ther. 8 (2), 47-55 (2015).</li><li>Rezvani, K., Rouce, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Application+of+Natural+Killer+Cell+Immunotherapy+for+the+Treatment+of+Cancer.">The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer.</a> Front Immunol. 6, 578 (2015).</li><li>Angelo, L. S., 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=Practical+NK+cell+phenotyping+and+variability+in+healthy+adults.">Practical NK cell phenotyping and variability in healthy adults.</a> Immunol Res. 62 (3), 341-356 (2015).</li><li>Zons, 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=Comparison+of+europium+and+chromium+release+assays:+cytotoxicity+in+healthy+individuals+and+patients+with+cervical+carcinoma.">Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma.</a> Clin Diagn Lab Immunol. 4 (2), 202-207 (1997).</li><li>Yovel, G., Shakhar, K., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+sex,+menstrual+cycle,+and+oral+contraceptives+on+the+number+and+activity+of+natural+killer+cells.">The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells.</a> Gynecol Oncol. 81 (2), 254-262 (2001).</li><li>Laue, T., 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=Altered+NK+cell+function+in+obese+healthy+humans.">Altered NK cell function in obese healthy humans.</a> BMC Obes. 2, 1 (2015).</li><li>Hazeldine, J., Lord, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+ageing+on+natural+killer+cell+function+and+potential+consequences+for+health+in+older+adults.">The impact of ageing on natural killer cell function and potential consequences for health in older adults.</a> Ageing Res Rev. 12 (4), 1069-1078 (2013).</li></ol>

Die Autoren erklären keine finanziellen Interessenkonflikte.

Die hier beschriebene Methode bietet eine einfache und kostengünstige Alternative zu den traditionellen 51Cr Release Assay zytotoxische NK-Zell-Aktivität zu bewerten. Diese Methode ist empfindlich, reproduzierbare und weniger zeitaufwändig als die bisherigen Standardmethoden wie CRA, und eignet sich für beide klinischen und Forschung Anwendungen.
Während des Tests mit insgesamt PBMCs und angereicherte NK-Zellen arbeitet, ist die Option PBMCs Zellpopulationen zu reinigen ohne die Notwendigkeit zu verwenden ein großer Vorteil beim Umgang mit Kleinmengen gesammelte Blut oder Paar oder schlechte Qualität Zellen von Patienten Proben. Dieser Assay nutzt nur CFSE- und tote Zelle Ausgrenzung Lebensfähigkeit Fleck. Betrachtet man diese beiden Parameter allein informiert prägnant und ausreichend zu diagnostischen Zwecken, mit dem zusätzlichen Vorteil der weitere Entschädigung in durchflusszytometrischen Analyse nicht erforderlich.
Diese Methode kann auch verwendet werden, grundlegende NK-Zell-Biologie zu studieren und testen neue NK Zelle gezielte Therapien. In dieser Hinsicht stellen die ständig wachsenden Möglichkeiten der fließen Cytometers wertvollen Bestandteil der Vielseitigkeit dieser Assay, zulassend multiparameter Analyse von NK-Zellen und ihre Tätigkeit nicht früher möglich mit Tests wie CRA. Alternative Zelle tracking und Lebensfähigkeit Farbstoffe, fluoreszierend in anderen Kanälen stehen die Ermittler Bedürfnisse zu erfüllen.
Dieses Protokoll ist für die Verwendung mit der prototypischen NK Zelle Ziel K562 Zelllinie optimiert. Es kann jedoch an alternativen Fangleinen Ziel Zelle mit unterschiedlichem Grad an Sensibilität für NK Zelle Zytotoxizität, wie HL-60, Daudi, U937 und Raji angepasst. In diesem Zusammenhang ist es wichtig zu beachten, dass die Konzentration von fluoreszierenden Farbstoff und Serum während Ziel Zelle Kennzeichnung verwendet möglicherweise für jede Zelllinie Ziel optimiert werden wie verschiedene Zelllinien das fluoreszierende Label anders und für jedes Durchflusszytometer nehmen könnte. Während es in der Regel zu vermeiden, mit Serum während Kennzeichnung empfohlen wird, entschieden wir uns zum leichten abschrecken mit 0,5 % des FBS aus zwei Gründen. Erstens hilft es Ziel Zelle Stress zu reduzieren und somit Hintergrund Flecken; Zweitens bringt es CFSE-Signal in den entsprechenden Bereich der Erkennung von unserem Durchflusszytometer ohne tägliche Einstellungen Anpassungen, so hilft in der Reproduzierbarkeit des Assays. Zusatzprotokolls Variationen beinhalten Tests verschiedener E:T Verhältnisse und Inkubationszeiten Assay unterschiedliche Aktivierung Bedingungen anzupassen. Die Dauer der Kokultur Effektor und Zielzellen, NK Zytotoxizität zu testen hat historisch von 4-16 h, reichten, obwohl längere Zeit tendenziell erhöhten spontane Freisetzung 9,16zur Folge haben. In unserer Methode könnte längere Inkubationszeit auch Verlust von Leuchtstofflampen, die Kennzeichnung durch die Zielzellen durch Tötung oder Verbreitung, führen, da diese Fluoreszenz-Farbstoffen auf Zellteilung verdünnt werden. So Inkubationen in das untere Ende des Zeitfensters werden in der Regel bevorzugt und sie über Nacht Inkubation 6,16,22nicht überschreiten.
Es ist wichtig, zur Kenntnis zu nehmen, von denen Variablen das Ergebnis dieses Tests beeinträchtigen können. Nach unserer Erfahrung sind die Zielzellen K562 besonders empfindlich auf Temperaturschwankungen. Daher K562-Zellen nicht gekühlt, sondern eher bei Raumtemperatur aufbewahrt oder platziert in einem befeuchteten CO2 Inkubator bei 37 ° C vor ihrer Verwendung. Aus dem gleichen Grund sollten alle Reagenzien zu den entsprechenden Temperaturen gebracht werden, wie im Protokoll angezeigt. Ein weiterer Parameter beeinflussen diese Zellen ist die Zentrifugation Geschwindigkeit, die reduziert werden sollte, um zellulären Stress zu minimieren. Darüber hinaus unbedingt begrenzen die Verzögerungszeit zwischen Effektor/Target-Vorbereitung und dem Beginn der Co Inkubation auf weniger als 30 min gewährleisten maximale Tötung Aktivität und assay Reproduzierbarkeit. Ebenso wie CFSE-Signal in der Regel robust ist, genaues pipettieren, richtige Mischung von Zellen und präzise Inkubationszeit Zeit beim etikettieren und abschrecken um Einheitlichkeit in der Färbung von entscheidender Bedeutung sind. Zu guter Letzt zytotoxischen Fähigkeit der NK-Zelle ist sehr variabel, auch bei gesunden Personen und ist beeinflusst durch eine Reihe von Faktoren, einschließlich der Entwicklungsstufe, Geschlecht, Alter und Gewicht 23,24,25. Zudem bis zu 30 % der gesunden Kontrollpersonen zeigen Sie eine signifikante Reduktion oder vollständiger Verlust der zytotoxischen Fähigkeit wenn länger als 24 Stunden nach der Blutabnahme getestet. Daher empfiehlt es sich, Verwendung frisch gereinigt PBMCs oder NK Zellen, wann immer möglich.

An erratum was issued for: <a href="https://www.jove.com/video/56191/a-flow-cytometry-based-cytotoxicity-assay-for-assessment-human-nk">A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity</a>. Figure 4 has been corrected to show background-corrected data.
Figure 4 was updated from:
<img alt="Figure 4" src="/files/ftp_upload/56191/56191fig4v1.jpg" />
to:
<img alt="Figure 4" src="/files/ftp_upload/56191/56191fig4v2.jpg" />

An erratum was issued for: <a href="https://www.jove.com/video/56191/a-flow-cytometry-based-cytotoxicity-assay-for-assessment-human-nk">A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity</a>. Figure 4 has been corrected to show background-corrected data.

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

Natürliche killer (NK)-Zellen sind eine anspruchsvolle Teilmenge der menschlichen angeborenen Lymphozyten kritisch beteiligt bei der Beseitigung von viral infizierte Zellen, transformierten Zellen und anderen pathogenen Bedrohungen 1,2. NK-Zellen lytische Granulat Haus zytotoxische Proteine, wie Perforin und Granzyme. Nach der Aktivierung NK-Zellen bilden ein komplexes Zusammenspiel mit ihren Zielen bekannt als immunologische Synapse, wobei diese Zytolyse Moleküle lokal freigesetzt werden, was direkte Ziel Zelle Lysis und Apoptose, zusammen mit Freisetzung von Zytokinen und Chemokin und letztlich in der Induktion von einem entzündlichen Zustand 1,3,4.
NK-Zell-Aktivierung beinhaltet eine komplexe Reihe von Aktivierung und hemmende Interaktionen zwischen NK Zell-Rezeptoren und Liganden auf der Oberfläche der Zielzellen, bilden ein streng regulierte System ausgedrückt. Einer der am besten untersuchten Mechanismen der NK-Zell-Aktivierung ist das "fehlende selbst". In der Tat, fehlende Erkennung der Klasse, die ich major Histocompatibility complex (MHC) oder human Leukocyte Antigen (HLA) Moleküle auf infiziert oder Zellen Trigger NK Zelle Zytotoxizität umgewandelt. Tumor und Virus-infizierten Zellen in der Regel Downregulate diese Antigene T zellvermittelte Immunität, so zum primären NK entkommen Zelle Ziele 1,3,4.
Bewertung der NK-Zell-Funktion ist in erster Linie in Degranulation oder Zytotoxizität kategorisiert Assays. Degranulation Assays, wie die Strömung durchflusszytometrischen Erkennung des Degranulation-assoziierten Markers CD107a, sind jedoch nur Richtwerte der NK-Zell-Aktivierung und nicht ihre ultimative Funktion, die direkte Tötung von Ziel Zellen 5,6,7,8. Daher hat diese Einschränkung Ermittler Zytotoxizität Assays als mehr erzählen und direktere Alternative geschenkt.
Die langjährigen "Goldstandard" für die Beurteilung der zellvermittelten zytotoxische Aktivität von T- und NK-Zellen ist die Chrom-Release-Assay (CRA). CRA beinhaltet radioaktiv Kennzeichnung von Zielzellen mit 51Cr und Bebrüten sie gemeinsam mit Effektorzellen. Dieser Assay ist durchdrungen von dem Grundsatz, dass diese Zelle lyse in der Version des proteingebundenen 51Cr in der Überstand ergibt sich die durch Gamma zählen gemessen werden kann. Dieser Assay, während wirksam, ist für eine Vielzahl von Gründen problematisch: hohe Materialkosten, Handhabung und Entsorgung von radioaktiven 51Cr, spontane Freisetzung von 51Cr und schwierigen Standardisierung - so dass es insgesamt unpraktisch 9,10.
Eine Reihe von nicht-radioaktiven Assays mit fluoreszierenden Kennzeichnung, Enzymfreisetzung und sogar Biolumineszenz, wurden seitdem als Alternativen zum CRA 11,12,13,14entwickelt. Wir beschreiben hier eine Flow Cytometry basierende Methode zur Messung der NK Zelle zytotoxische Aktivität auf K562 Zielzellen, einfache, sensitive und reproduzierbar ist. K562-Zellen sind eine menschliche Erythroleukemic Zelle Linie mit reduzierten Expression von HLA Klasse I und erhöhten Ausdruck von Liganden für activatory NK-Rezeptoren, wodurch sie besonders anfällig für NK zellvermittelte Zytotoxizität 15. In diesem Assay K562 Zellen vorab mit Carboxyfluorescein Diacetat Succinimidyl Ester (CFSE-) gekennzeichnet sind und Co kultiviert in verschiedenen Verhältnissen mit entweder peripheren mononukleären Blutzellen (PBMCs) oder NK-Zellen 1gereinigt. CFSE ist eine stabile, Proteinbindung Fluoreszenzfarbstoff, der Diskriminierung von Zielzellen von Effektor NK Zellen 16,17erlaubt. Nach der Co Inkubation dient ein Nukleinsäure-Fleck, speziell durchdringt die Membran von abgestorbenen Zellen, getöteten Zielzellen identifizieren (siehe Tabelle der Materialien). Die Proben werden dann auf einem Durchflusszytometer zu bestimmen, den Anteil der Toten (d. h. Fleck +) erworben CFSE-+ Zielzellen.
Dieser Test kann verwendet werden als diagnostische Reihenuntersuchung für monogene Mängel beeinträchtigen die NK Zellkompartiment, davon gibt es etwa 30 bekannte Mängel verursacht entweder funktionelle oder klassische NK-Zelle-Mangel, und bei primären oder sekundären Hemophagocytic Lymphohistiocytosis. Es ist auch sinnvoll, NK-Zell-Aktivität bei Patienten mit wiederkehrenden, schweren viralen Herpesinfektionen, Immunrekonstitution nach hämatopoetischer Stammzelltransplantation oder Post immunmodulatorische Therapie 18,19,20, bewerten und für eine Vielzahl von Anwendungen in der grundlegenden Forschung zu untersuchen.

<table><tbody><tr><td>Phosphate-buffered Saline (1x, w/o Ca2+ and Mg2+)</td><td>Corning (Cellgro)</td><td>21-040-CM</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>BP337-100</td><td></td></tr><tr><td>RPMI 1640 Media</td><td>Corning (Cellgro)</td><td>10-040-CV</td><td></td></tr><tr><td>Heat-inactivated Fetal Bovine Serum</td><td>Omega Scientific</td><td>FB-02</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140-163</td><td>Stock solution at 10,000 U/mL</td></tr><tr><td>IL-2</td><td>R&amp;amp;D Systems</td><td>202-IL-050</td><td>Lyophilized from a 0.2 &amp;mu;m filtered solution in Acetonitrile and TFA with BSA as a carrier protein. Reconstitute with 500 ul at 100 &amp;mu;g/mL in sterile 100 mM Acetic Acid containing at least 0.1% bovine serum albumin (2.1x10E6 IU/ml)</td></tr><tr><td>K562 Cells</td><td>ATCC</td><td>CCL-243</td><td>Cancer cell line&amp;nbsp;</td></tr><tr><td>T-75 cell culture flasks</td><td>Corning</td><td>431464</td><td></td></tr><tr><td>CFSE cell proliferation kit</td><td>Life Technologies (CellTrace)</td><td>C34554</td><td>Reconstitute I vial with 18 ul DMSO to prepare a 5mM stock solution. Do not freeze/thaw.</td></tr><tr><td>Sytox Red</td><td>Life Technologies</td><td>S34859</td><td>Stock solution is provided at 5 &amp;mu;M in 1 mL DMSO. The DMSO solution may be subjected to multiple freeze-thaw cycles without reagent degradation.</td></tr><tr><td>Sodium/lithium heparin blood collection tubes</td><td>BD</td><td>02-687-95</td><td></td></tr><tr><td>U-bottom 96-well plate</td><td>Corning</td><td>CLS3897</td><td></td></tr><tr><td>Serological pipettes</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>Polystyrene round-bottom tubes (5mL)</td><td>BD Falcon</td><td>14959-5</td><td></td></tr><tr><td>50 mL polypropylene conical tube</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>15 mL polypropylene conical tube</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>Reagent reservoir</td><td>USA Scientific</td><td>2321-2230</td><td></td></tr><tr><td>Human NK cell enrichment cocktail</td><td>StemCell Technologies (RosetteSep)</td><td>15065</td><td></td></tr></tbody></table>

a flow cytometry-based cytotoxicity assay for the assessment of human nk cell activity

Innerhalb des angeborenen Immunsystems spielen Effektor-Lymphozyten, die sogenannten natürlichen killer (NK)-Zellen eine wesentliche Rolle in der Wirt Verteidigung gegen anomale Zellen, insbesondere Beseitigung Tumor und viral infizierte Zellen. Ca. 30 bekannten monogenen Defekte, zusammen mit einer Vielzahl von anderen pathologischen Zuständen verursachen entweder funktionelle oder klassische NK Zelle Mangel, manifestiert sich in reduziert oder fehlend zytotoxische Aktivität. In der Vergangenheit wurde Zytotoxizität mit radioaktiven Methoden untersucht die mühsam, teuer und potenziell gefährlich sind. Dieser Artikel beschreibt eine optimierte, klinisch einsetzbaren Flow Cytometry basierende Methode um zytotoxische NK-Zell-Aktivität zu quantifizieren. In diesem Test sind peripheren mononukleären Zellen (PBMCs) oder gereinigte NK Zelle Vorbereitungen Co inkubierten bei unterschiedlichen Verhältnissen eine Ziel-Tumor-Zell-Linie bekannt, empfindlich gegen NK zellvermittelte Zytotoxizität (NKCC) sein. Die Zielzellen sind bereits beschriftet mit einem Fluoreszenzfarbstoff ermöglicht ihre Diskriminierung aus den Effektorzellen (NK-Zellen). Nach der Inkubationszeit sind getöteten Zielzellen durch eine Nukleinsäure-Fleck, identifiziert, die speziell die abgestorbenen Zellen durchdringt. Diese Methode ist geeignet, um sowohl Diagnose- und Anwendungen in der Forschung und, Dank an die Multiparameter-Fähigkeiten der Durchflusszytometrie, hat den zusätzlichen Vorteil der potenziell ermöglicht eine tiefere Analyse der NK Zelle Phänotyp und Funktion.

Vor der Inbetriebnahme der Assay empfiehlt es sehr, dass NK Zellinhalt in der Effektor Bevölkerung Wahl beurteilt werden. Abbildung 1 zeigt eine typische CD56 Färbung vor (hellblau) und nach (rot) NK Zelle Bereicherung. NK-Zellen sollte bestehen aus bis zu 15 % der PBMCs und mindestens 80 % rein nach der Bereicherung.
Durchflusszytometrischen Analyse in diesem Assay umfasst Erkennung von zwei Parameter: CFSE, nachweisbar in den gleichen Kanal wie FITC; und eine tote Zelle Fleck, im gleichen Kanal wie APC (Abb. 2AB) nachweisbar. Nach der Datenerfassung wird die gating-Strategie in Abbildung 2 verwendet, um Daten zu analysieren. Toten (z.B. tote Zelle Fleck +) K562-Ziel-Zellen werden aus der CFSE-+ Bevölkerung, für die % der getöteten Zellen innerhalb der Zielpopulation angespritzt.
Kontrolle Bedingungen sind entscheidend für die Gewährleistung der Wirksamkeit des Tests selbst und seiner Einzelkomponenten. In Reihenfolge für den Test als gültig betrachtet werden, die folgenden erwartet werden sollte, bei der Kontrolle (6-8): Zellen nur (Zustand 6) sollte im Zelltod führen &#60; 15 % (Abb. 3A). Kein CFSE--Signal sollte erkannt werden, für Effektor nur (Zustand 7 Zellen), und Zelltod sollte &#60; 5 % (Abb. 3 b). Tween-vermittelten Tötung von Zielzellen (Bedingung 8), Zelltod sollte &#62; 85 % (Abbildung 3).
Ein Patient mit Defekt in der NK-Zell-Funktion wird voraussichtlich Tötung Aktivität in den meisten verringert haben oder alle Kennzahlen im Vergleich zu einem gesunden Menschen getestet. Die parallele von gesunden Spendern und Patientenzellen in Abbildung 4 zeigt differential, deutliche Reduzierung Ziel Zelltod mit abnehmender Effektor-Zielquote Zelle.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56191/56191fig1.jpg"> 
Abbildung 1: Bewertung der NK Zelleninhalt innerhalb der Bevölkerung Effektor. Repräsentative Histogramm nach dem Färben total PBMCs (hellblau) oder NK-Zellen (rot) für CD56 gereinigt. <a href="//ecsource.jove.com/files/ftp_upload/56191/56191fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56191/56191fig2.jpg"> 
Abbildung 2: Strategie zur Erkennung von Zielzellen Gating. A) das erste Tor befindet sich in einem FITC-A/SSC-A-Grundstück. K562-Zellen werden als CFSE-+ Veranstaltungen angespritzt. B) Tote K562 Zellen (d. h. positiv für tote Zelle färben) werden in der nachfolgenden APC-A/SSC-A Handlung innerhalb der CFSE-+ Zelle Zielpopulation angespritzt. <a href="//ecsource.jove.com/files/ftp_upload/56191/56191fig2large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56191/56191fig3.jpg"> 
Abbildung 3: repräsentative Daten für Kontrolle Bedingungen. (A) Anteil der Toten Zelle Fleck + K562 Zellen innerhalb des CFSE-+ Gates (gating Strategie wie in Abbildung 2) Zustand 6 (nur Zielzellen) - negativ-Kontrolle für K562 Zelltod. B) CFSE- und tote Zelle färben-Erkennung in Bedingung 7 (Effektor Zellen nur - total PBMCs). C) Anteil der Toten Zelle Fleck + K562 Zellen innerhalb des CFSE-+ Gates (gating Strategie wie in Abbildung 2) Zustand 8 (Zielzellen + Tween) - Positivkontrolle für K562 Zelltod. <a href="//ecsource.jove.com/files/ftp_upload/56191/56191fig3large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56191/56191fig4.jpg"> 
Abbildung 4: repräsentative Daten für den Verwässerungseffekt der verschiedenen Effektor Zellen-Zielquote. Die Effektor: Ziel-Zelle-Verhältnisse getestet sind wie folgt: 50: 1, 25: 1; 12,5: 1; 6.25:1. Daten sind Hintergrund (Bedingung #6)-entfernt. <a href="//ecsource.jove.com/files/ftp_upload/56191/56191fig4large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Probe</td>
			<td>Zustand</td>
			<td>Effektor (E): Ziel (T)</td>
		</tr>
		<tr>
			<td rowspan="8">PBMCs</td>
			<td>1 - Positivkontrolle für NK Zelle Zytotoxizität</td>
			<td>50:1 + IL-2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>50: 1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>25: 1</td>
		</tr>
		<tr>
			<td>4</td>
			<td>12,5: 1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>6.25: 1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>T nur</td>
		</tr>
		<tr>
			<td>7 - negativ-Kontrolle für K562 Tod</td>
			<td>E nur</td>
		</tr>
		<tr>
			<td>8 - Positivkontrolle für K562 Tod</td>
			<td>T + Tween</td>
		</tr>
		<tr>
			<td rowspan="8">NK-Zellen (gereinigt)</td>
			<td>1 - Positivkontrolle für NK Zelle Zytotoxizität</td>
			<td>5:1 + IL-2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>5: 1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>2,5: 1</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1,25: 1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>0,625: 1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>T nur</td>
		</tr>
		<tr>
			<td>7 - negativ-Kontrolle für K562 Tod</td>
			<td>E nur</td>
		</tr>
		<tr>
			<td>8 - Positivkontrolle für K562 Tod</td>
			<td>T + Tween</td>
		</tr>
	</tbody>
</table>
Tabelle 1

Watch this Scientific Journal Video about A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity at JoVE.com

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

Eine Flow Cytometry basierende Methode quantitativ bestimmen die zytotoxische Aktivität des menschlichen natürliche Killerzellen wird hier angezeigt.

Ein Flow Cytometry-basierte Zytotoxizität Assay für die Beurteilung der menschlichen NK-Zell-Aktivität

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

a-flow-cytometry-based-cytotoxicity-assay-for-assessment-human-nk

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.7K Aufrufe.  University of California, Los Angeles. Eine Flow Cytometry basierende Methode quantitativ bestimmen die zytotoxische Aktivität des menschlichen natürliche Killerzellen wird hier angezeigt.

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, Reinigung und Functional Assessment of Human peripheren Blut NK-Zellen

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Forschung

Immunologie und Infektion

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.

Herstellung und Verwendung von HIV-1 infizierten primären CD4 + T-Zellen als Zielzellen in der natürlichen Killerzellen Zytotoxische 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 ...

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.

Durchflusszytometrie-basierte Assay zur Überwachung von NK-Zell-Funktionen

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

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.

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.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

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

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

Ein platebasierter Cytotoxicity Assay zur Bewertung der Rate Placental Natural Killer Cell Cytolytic Funktion

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

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.

Bewertung menschlicher natürlicher Killerzellereignisse, die durch fcγRIIIa Engagement in Gegenwart therapeutischer Antikörper angetrieben werden

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

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.

Messung der natürlichen Killerzell-vermittelten Zytotoxizität und Migration im Kontext von leberassoziierten Tumorzellen

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 ...

Medizin

Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

Source: Chava, S. et al., <a href="https://app.jove.com/v/60714">Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells.</a> J. Vis. Exp. (2020)
This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.

Calcein-Acetoxymethyl-Färbung zur Bewertung der natürlichen Killerzell-vermittelten Zytotoxizität gegenüber Krebszellen

Source: Chava, S. et al., Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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.