Nos gustaría agradecer a Jill Narciso, centro de inmunogenética de UCLA, por su asistencia con la preparación del manuscrito.

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>

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Los autores no declaran conflictos de interés financiero.

El método descrito aquí proporciona una alternativa sencilla y rentable para el ensayo de lanzamiento tradicionales 51Cr para evaluar la actividad citotóxica de células NK. Este método es sensible, reproducible y menos desperdiciador de tiempo que los anteriores métodos estándar, como CRA y puede ser utilizado para ambas clínicas y aplicaciones de investigación.
Mientras que el ensayo trabaja con total PBMCs y enriquecida de las células NK, la opción de usar PBMCs sin la ...

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

Células de naturales del asesinas (NK) son un subconjunto sofisticado de linfocitos innatos humanos críticamente implicados en la eliminación de las células viralmente infectadas, las células transformadas y otras amenazas patógenas 1,2. Casa de gránulos líticos de la célula NK proteínas citotóxicas, como perforin y granzymes. Tras la activación, las células NK forman una interacción compleja con sus objetivos conocidos como sinapsis inmunológica, por el que estas moléculas citolíticas se liberan localmente, dando por resultado la lisis de la célula objetivo directo y apoptosis, junto con liberación de citoquinas y quimioquinas y en última instancia en la inducción de un inflamatorio estado 1,3,4.
Activación de la célula NK involucra una compleja cadena de activación y las interacciones inhibitorias entre NK célula receptores y ligandos expresadas en la superficie de las células de la blanco, formando un sistema fuertemente regulado. Uno de los mecanismos más estudiados de la activación de células NK es la "falta". De hecho, falta de detección de clase en que mayor complejo de histocompatibilidad (MHC), o moléculas de leucocito humano antígeno (HLA), infectados o transformado citotoxicidad celular de células desencadenantes NK. Tumorales y células infectadas por virus generalmente regular a la baja estos antígenos para escapar de la inmunidad mediada por células T, convirtiéndose en principal NK célula objetivos 1,3,4.
Evaluación de la función de la célula NK se categoriza principalmente en degranulación o citotoxicidad ensayos. Sin embargo, ensayos de degranulación, como detección de citometría de flujo del degranulation asociados marcador CD107a, sólo son indicativos de la activación de células NK y no de su función final, la muerte directa de las células objetivo 5,6,7,8. Por lo tanto, esta limitación ha dibujado los investigadores ensayos de citotoxicidad como alternativa más directa y más revelador.
El largo plazo "gold standard" para evaluar la actividad citotóxica mediada por células de las células T y NK es el ensayo de liberación de cromo (CRA). CRA implica radiactivo etiquetado de células diana con 51Cr y co los incuban con las células efectoras. Este ensayo se empapa en el principio que lisis celular resulta en la liberación de proteína determinada 51Cr en el sobrenadante, que puede ser medido contando gamma. Este ensayo, al mismo tiempo eficaz, es problemático para una variedad de razones: altos costos de material, manejo y disposición de radiactivo 51Cr, lanzamiento espontáneo de 51Cr y difícil estandarización - lo que en conjunto práctico 9,10.
Un número de ensayos no radiactivo, con etiquetado fluorescente, liberación de enzima e incluso bioluminiscencia, desde entonces se han desarrollado como alternativas a la CRA 11,12,13,14. Aquí describimos un método basado en la citometría de flujo para medición de NK célula actividad citotóxica en las células K562 objetivo simple, sensible y reproducible. Células K562 son una células erythroleukemic humana la línea con menor expresión de HLA clase I y una mayor expresión de ligandos para receptores NK activatory, que los hace particularmente susceptibles a NK Citotoxicidad mediada por células 15. En este ensayo, las células K562 están marcadas previamente con carboxyfluorescein diacetato succinimidyl éster (CFSE) y cultivan conjuntamente en diferentes proporciones con cualquiera células mononucleares de sangre periférica (PBMCs) o purificaron de células NK 1. CFSE es un colorante fluorescente estable, proteínas de Unión que permite la discriminación de las células de la blanco del efectoras NK células 16,17. Después de la incubación Co, una mancha de ácido nucleico, específicamente impregnando la membrana de las células muertas, se utiliza para identificar las células de la blanco matado (véase tabla de materiales). Las muestras entonces se adquieren en un citómetro de flujo para determinar el porcentaje de muertos (es decir, mancha +) CFSE + células diana.
Este ensayo puede utilizarse como una investigación rutinaria de diagnóstico para defectos monogénicas, que afectan el compartimiento de células NK, que son aproximadamente 30 defectos conocidos que causan deficiencia de células NK ya sea funcional o clásica, y para el lymphohistiocytosis hemophagocytic primario o secundario. También es útil para investigar la actividad de células NK en pacientes con infecciones virales herpes recurrente, grave, para evaluar la reconstitución inmune tras el trasplante de células hematopoyéticas o post inmunomoduladores terapia 18,19,20y de una serie de aplicaciones de la investigación básica.

<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&#38;D Systems</td>
			<td>202-IL-050</td>
			<td>Lyophilized from a 0.2 &#956;m filtered solution in Acetonitrile and TFA with BSA as a carrier protein. Reconstitute with 500 ul at 100 &#956;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&#160;</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 &#956;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

Dentro del sistema inmune innato, los linfocitos efectores conocidos como células de naturales del asesinas (NK) juegan un papel esencial en la defensa del huésped contra las células aberrantes, especialmente eliminando tumoral y viralmente infectadas las células. Aproximadamente 30 defectos monogénicas conocidos, junto con una serie de otras condiciones patológicas, deficiencia funcional o clásico NK célula, que se manifiesta en reducción o ausencia de actividad citotóxica. Históricamente, se ha investigado la citotoxicidad con métodos radiactivos, que son engorrosos, costosos y potencialmente peligrosos. Este artículo describe un método basado en la citometría de flujo aerodinámico, clínicamente aplicable para cuantificar la actividad citotóxica de células NK. En este ensayo, las células mononucleares de sangre periférica (PBMCs) o preparaciones de células NK purificadas son co se incubó a relaciones diferentes con una línea de células de tumor objetivo sabida que es sensible a la citotoxicidad mediada por células NK (NKCC). Las células diana son las marcadas con un colorante fluorescente para permitir la discriminación de las células efectoras (células NK). Después del período de incubación, las células diana mataron son identificadas por una mancha de ácido nucleico, que impregna específicamente células muertas. Este método es susceptible de diagnóstico y aplicaciones de investigación y, gracias a las capacidades de múltiples parámetros de la citometría de flujo, tiene la ventaja añadida de que potencialmente permite un análisis más profundo del fenotipo de la célula NK y la función.

Antes de establecer el ensayo, se recomienda evaluar contenido de células NK en la población de efector de elección. La figura 1 muestra una típica coloración CD56 antes (azul claro) y después de enriquecimiento de células NK (rojo). Las células NK constituyen hasta un 15% de PBMCs y deben ser por lo menos 80% de pureza después de enriquecimiento.
El análisis cytometric del flujo en este e...

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

Aquí se muestra un método basado en la citometría de flujo para determinar cuantitativamente la actividad citotóxica de las células de asesino naturales humanas.

Un análisis de citotoxicidad basados en la citometría de flujo para la evaluación de la actividad de las células NK humanas

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

Research

JoVE Journal

Immunology and Infection

23.6K vistas.  University of California, Los Angeles. Aquí se muestra un método basado en la citometría de flujo para determinar cuantitativamente la actividad citotóxica de las células de asesino naturales humanas.

Video: 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.

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

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

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

Investigación

Inmunología e Infección

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las células T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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 ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Evaluación rápida y específica de la peroxidasa Actividad de halogenación en muestras de sangre enriquecida en leucocitos

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

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.

Ensayo de citometría de flujo de base para la supervisión de funciones de las células NK

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

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La evaluación de la función retiniana Microglial fagocítica<em&gt; En Vivo</em&gt; El uso de un ensayo basado en citometría de flujo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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.

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

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 ...

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.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

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

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...