Name: single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry
Uploaded: 2018-06-26T13:30:00
Description: Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

Nos gustaría agradecer a Aimee Pugh-Bernard por su entrada intelectual y comentarios útiles. Este trabajo fue apoyado por el Premio de investigación biomédica Boettcher Foundation Webb Waring y Premio número K23-1K23AR070897 de los NIH a Elena W.Y. Hsieh. Ella también fue apoyada por número de concesión HD000850 K12 del Eunice Kennedy Shriver Instituto Nacional de salud infantil y desarrollo humano y la Lucile Packard Foundation para la salud de los niños, Stanford CTSA UL1 TR001085 e investigación en salud infantil Instituto de Stanford University.

Todos los métodos aquí descritos han sido aprobados por el Colorado múltiples institucional de junta (COMIRB) de la Universidad de Colorado. Todos los procedimientos descritos a continuación deben realizarse en una campana de cultivo estériles para tejidos, salvo estipulación en contrario, con las puntas de pipeta filtrada, y filtraron todos los reactivos.
1. preparación de reactivos para el procesamiento de la sangre periférica
<ol><li>Preparar el ruxolitinib alícuotas stock (10-15...

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Aquí describimos una novela, unicelular, enfoque proteómico para evaluar simultáneamente múltiples tipos de células inmunes y detectar sus diferentes perturbaciones de citoquinas en el ambiente de paciente "patógenos" plasma de la sangre periférica circulante factores específicos. Este método emplea sangre periférica como el vehículo analítico y la citometría de masas como herramienta para la evaluación de anormalidades fenotípicas y funcionales celulares inmunes. El método es fácilmente aplicable a huma...

Las enfermedades autoinmunes son una causa importante de morbilidad y mortalidad que afecta a 3-8% de la población. En los Estados Unidos, trastornos autoinmunes se encuentran entre las principales causas de muerte entre las mujeres jóvenes y de mediana edad (edad < 65 años)1,2. Enfermedades autoinmunes se caracterizan por la presentación clínica heterogénea y los diversos procesos inmunológicos subyacentes. El espectro de la heterogeneidad del bien es representado a través de diferentes trastornos, como la participación conjunta en la artritis reumatoide (ar) y enfermedades neurológicas en la esclerosis múltiple (MS). Sin embargo, este nivel de heterogeneidad se ejemplifica también dentro de un desorden único, como el lupus eritematoso sistémico (les): algunos pacientes pueden presentar con patología renal, mientras que otros sufren de implicación hematológica o común3.
La inmunopatogénesis subyacente en los trastornos autoinmunitarios refleja la heterogeneidad clínica, con auto y hyper-activación de múltiples subconjuntos de la célula inmune innata y adaptativa y producción de citoquinas desregulación concomitante. Mientras que las citoquinas juegan un papel fundamental en la patogenia de la enfermedad autoinmune, entender su papel específico en el mecanismo de la enfermedad ha demostrado para ser un reto. Citocinas se caracterizan por pleiotropía (una citocina puede tener múltiples efectos en diferentes tipos de células), redundancia (varias citocinas pueden tener el mismo efecto), dualidad (una citocina puede tener efectos pro o antiinflamatoria bajo diversas condiciones), y plasticidad (citoquinas pueden ser moldeadas en un papel diferente de su "original", según el medio ambiente)4,5,6. Por lo tanto, métodos de nivel de la población no pueden distinguir heterogéneas respuestas celulares a la misma «medio de cytokine". Asimismo, diseños de los estudios que se centran en un aspecto específico del sistema inmune (un solo tipo de células o citocinas), no ofrecen una valoración global de todos los elementos que intervienen en complejas enfermedades autoinmunes. Mientras que estudios de sueros de pacientes han producido penetraciones importantes en autoinmunidad, no proporcionan la fuente celular específica de las citocinas de desajuste detectadas.
Recientemente, hemos desarrollado un enfoque proteómico unicelular para evaluar simultáneamente múltiples tipos de células inmunes y detectar sus diferentes perturbaciones de citoquinas en el medio de pacientes "patógenos" plasma de la sangre periférica circulante factores específicos. El flujo de trabajo descrito aquí se caracteriza por el uso de muestras de sangre periférica intacta, a diferencia de las células mononucleares aislados de sangre periférica (PBMCs). Sangre periférica representa el vehículo más fisiológicamente relevante para el estudio de enfermedad mediada inmune sistémica, incluyendo 1) no-células mononucleares de sangre a menudo implicados en enfermedad autoinmune (es decir, neutrófilos, plaquetas) y plasma 2) circulación de factores, tales como ácidos nucleicos, complejos inmunes y citocinas, que tienen funciones de activación inmunes. Para capturar la "intrínseca patógenos" desequilibrada producción de citoquinas, las muestras son procesadas inmediatamente después del sorteo de la sangre (T0, tiempo cero) y después de 6 h de incubación a 37 ° C (temperatura corporal fisiológica) con un transporte de la proteína periférica de la sangre inhibidor en la ausencia de cualquier condición estimulante exógeno (T6, tiempo 6 h), para detectar la producción de citoquinas (acumulación, T6-T0) que refleje el estado de "intrínseco" de la enfermedad (figura 1). Para el estudio de procesos de desregulación que reflejan sobre o debajo-activación de la señalización vías implicadas en las respuestas inmunológicas pertinentes a la enfermedad, las muestras de sangre periférica son tratados (6 h de incubación a 37 ° C con un inhibidor del transporte de la proteína) con una exógena estimular la condición que refleja la patogénesis de la enfermedad, como los agonistas de Toll-Like-Receptor (TLR) en el caso de SLE (T6 + R848, tiempo 6 h con 1 μg/mL R848), para detectar la producción de citoquinas que reflejaría una respuesta a los ácidos nucleicos (comparando T0 vs T6 vs T6 + R848, figura 1). Para estudiar los efectos inmunomoduladores de la terapéutica disponible ex vivo, lo que se refiere a los procesos de desregulación inmune precisa para ciertos pacientes, muestras de sangre periférica son tratadas con un inhibidor JAK en la correspondiente terapéutico concentración (aquí, 5 uM ruxolitinib; T6 + 5R, tiempo 6 h con 5 uM ruxolitinib), para detectar cambios en el estado de enfermedad "intrínseca" en respuesta a la droga (T0 vs T6 vs T6 + 5R, figura 1). Un inhibidor JAK fue elegido para este estudio porque inhibidores de JAK se han demostrado para tener éxito en el tratamiento de trastornos autoinmunes como la RA
Evaluar simultáneamente los procesos de estabilización descritos en varios subconjuntos de células inmunitarias, muestras de sangre periférica de SLE pacientes y controles sanos se procesada como se describió anteriormente y se analizaron por citometría de masas. Citometría de masas, también denominada citometría-tiempo de vuelo, ofrece análisis unicelular de parámetros más de 40 sin problemas de espectral solapan de8,7,9. Esta técnica utiliza isótopos metal de tierras raras en forma de iones metálicos solubles como etiquetas a los anticuerpos, en lugar de fluoróforos10. Detalles adicionales sobre la plataforma tecnológica de citometría de masas (es decir, ajuste y calibración, adquisición de muestra) se pueden encontrar en Leipold et al. y McCarthy et al. 11 , 12 la alta dimensionalidad de citometría de masas permite la medida simultánea de múltiples citocinas en subconjuntos de la célula inmune innata y adaptativa con granularidad unicelular (Tabla de materiales).
Parámetros clínicos y de laboratorio convencionales actuales no son a menudo sensible o lo suficientemente específicos para la detección de actividad de la enfermedad actual o la respuesta a los inmunomoduladores específicos13, reflejando la necesidad de delimitar mejor la inmune subyacente cambios que impulsan las llamaradas. Dada la omnipresencia del cytokine dysregulation en enfermedad autoinmune, tiene una plétora de enfoques de tratamiento que utilizan anticuerpos o inhibidores moleculares pequeños contra citoquinas o señalización de las proteínas implicadas en la regulación de la producción de citoquinas recientemente surgido. En su formato básico, el enfoque analítico de sangre periférica descrito aquí proporciona una plataforma para identificar subconjuntos de la célula altera específico para cada paciente y su producción anormal de citocinas en la enfermedad autoinmune con manifestaciones sistémicas. Esta metodología permite la personalización de opciones terapéuticas como citoquinas altera específico pueden ser identificados, y tratamiento específico pueden ser opciones prueba ex vivo para evaluar su capacidad de immunomodulate el paciente enfermedad específica proceso.

<table>
	<tbody>
		<tr>
			<td>Ruxolitinib</td>
			<td>Santa Cruz</td>
			<td>SC-364729A</td>
			<td>Stock Conc: 10 mM; Final Conc: 5 &#956;M</td>
		</tr>
		<tr>
			<td>R848</td>
			<td>Invivogen</td>
			<td>tlrl-r848-5</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>LPS-EK</td>
			<td>Invivogen</td>
			<td>tlrl-eklps</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyse/Fix Buffer</td>
			<td>BD biosciences</td>
			<td>558049</td>
			<td>Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>&#160;BD Phosflow perm/wash buffer I</td>
			<td>BD biosciences</td>
			<td>557885</td>
			<td>Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
			<td>Stock Conc: &#62;99.9%; Final Conc: 0.0002</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (PTI)</td>
			<td>eBiosciences</td>
			<td>00-4980-93</td>
			<td>Stock Conc: 500X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>DNA Intercalator</td>
			<td>Fluidigm</td>
			<td>201192B</td>
			<td>Stock Conc: 500 &#956;M; Final Conc: 0.1 &#956;M</td>
		</tr>
		<tr>
			<td>Cell Staining Media (CSM)</td>
			<td></td>
			<td></td>
			<td>PBS + 0.5% BSA, 0.02% NaN3</td>
		</tr>
		<tr>
			<td>MaxPar Barcode Perm Buffer</td>
			<td>Fluidigm</td>
			<td>201057</td>
			<td>Stock Conc: 10X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>20-plex Pd Barcode Set</td>
			<td>Fluidigm</td>
			<td>S0014</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>EQ TM Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td>Stock Conc: 10X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>16% MeOH-free Formaldehyde Solution</td>
			<td>Thermo</td>
			<td>28908</td>
			<td>Stock Conc: 16% (w/v); Final Conc: 1.6% (w/v)</td>
		</tr>
		<tr>
			<td>Sterile round bottom polystyrene tubes</td>
			<td>VWR</td>
			<td>60818-496</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>Polypropylene cluster tubes</td>
			<td>Light Labs</td>
			<td>A-9001</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>Helios CyTOF instrument</td>
			<td>Fluidigm</td>
			<td>Helios</td>
			<td>All solutions to be used in CyTOF analysis need to be free of metal contamination. ddH2O is used in the preparation of any solutions should have a resistivity of at least 18.0 M&#937;.cm. ddH2O and any self-prepared solutions should be stored in new plastic or glass bottles that have never been autoclaved.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies used for Mass Cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surface markers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD1c</td>
			<td>Biolegend</td>
			<td>L161</td>
			<td>Mass: 161</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD</td>
			<td>UCHT1</td>
			<td>Mass: 144</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>SK3</td>
			<td>Mass: 174</td>
		</tr>
		<tr>
			<td>CD7</td>
			<td>BD</td>
			<td>M-T701</td>
			<td>Mass: 149</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Biolegend</td>
			<td>SK1</td>
			<td>Mass: 142</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Fluidigm</td>
			<td>ICRF44</td>
			<td>Mass: 209</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>BD</td>
			<td>B-ly6</td>
			<td>Mass: 152</td>
		</tr>
		<tr>
			<td>CD15</td>
			<td>BD</td>
			<td>HI98</td>
			<td>Mass: 115</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>Biolegend</td>
			<td>M5E2</td>
			<td>Mass: 154</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>eBioscience/Thermo</td>
			<td>B73.1</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Santa Cruz</td>
			<td>SJ25C1</td>
			<td>Mass: 163</td>
		</tr>
		<tr>
			<td>CD21</td>
			<td>Biolegend</td>
			<td>Bu32</td>
			<td>Mass: 141</td>
		</tr>
		<tr>
			<td>CD27</td>
			<td>BD</td>
			<td>L128</td>
			<td>Mass: 155</td>
		</tr>
		<tr>
			<td>CD38</td>
			<td>Fluidigm</td>
			<td>HIT2</td>
			<td>Mass: 172</td>
		</tr>
		<tr>
			<td>CD45 total</td>
			<td>Biolegend</td>
			<td>HI30</td>
			<td>Mass: 89</td>
		</tr>
		<tr>
			<td>CD45RA</td>
			<td>Biolegend</td>
			<td>HI100</td>
			<td>Mass: 153</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Miltenyi</td>
			<td>REA196</td>
			<td>Mass: 168</td>
		</tr>
		<tr>
			<td>CD66</td>
			<td>BD</td>
			<td>B1.1/CD66</td>
			<td>Mass: 113</td>
		</tr>
		<tr>
			<td>CD86</td>
			<td>Fluidigm</td>
			<td>IT2.2</td>
			<td>Mass: 150</td>
		</tr>
		<tr>
			<td>CD123</td>
			<td>Fluidigm</td>
			<td>6H6</td>
			<td>Mass: 143</td>
		</tr>
		<tr>
			<td>CD278/ICOS</td>
			<td>Biolegend</td>
			<td>C398.4A</td>
			<td>Mass: 156</td>
		</tr>
		<tr>
			<td>CD179/PD1</td>
			<td>Biolegend</td>
			<td>EH12.2H7</td>
			<td>Mass: 162</td>
		</tr>
		<tr>
			<td>IgD</td>
			<td>Biolegend</td>
			<td>IA6-2</td>
			<td>Mass: 146</td>
		</tr>
		<tr>
			<td>IgM</td>
			<td>Biolegend</td>
			<td>MHM-88</td>
			<td>Mass: 151</td>
		</tr>
		<tr>
			<td>CXCR5</td>
			<td>BD</td>
			<td>RF8B2</td>
			<td>Mass: 173</td>
		</tr>
		<tr>
			<td>HLADR</td>
			<td>Biolegend</td>
			<td>L243</td>
			<td>Mass: 167</td>
		</tr>
		<tr>
			<td>Cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#945;</td>
			<td>Biolegend</td>
			<td>364-3B3-14</td>
			<td>Mass: 147</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>Biolegend&#160;</td>
			<td>H1b-98</td>
			<td>Mass: 169</td>
		</tr>
		<tr>
			<td>IL-1RA</td>
			<td>Santa Cruz&#160;</td>
			<td>AS17</td>
			<td>Mass: 157</td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>Biolegend</td>
			<td>MQ2-13A5</td>
			<td>Mass: 164</td>
		</tr>
		<tr>
			<td>IL-8</td>
			<td>BD</td>
			<td>E8N1</td>
			<td>Mass: 160</td>
		</tr>
		<tr>
			<td>IL-12/IL-23p40</td>
			<td>Biolegend&#160;</td>
			<td>C8.6</td>
			<td>Mass: 171</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>Biolegend&#160;</td>
			<td>BL168</td>
			<td>Mass: 148</td>
		</tr>
		<tr>
			<td>IL23p19</td>
			<td>eBioscience/Thermo</td>
			<td>23dcdp</td>
			<td>Mass: 176</td>
		</tr>
		<tr>
			<td>MIP1&#946;</td>
			<td>BD</td>
			<td>D21-1351</td>
			<td>Mass: 158</td>
		</tr>
		<tr>
			<td>MCP1</td>
			<td>BD</td>
			<td>5D3-F7</td>
			<td>Mass: 170</td>
		</tr>
		<tr>
			<td>IFN&#945;</td>
			<td>Miltenyi&#160;</td>
			<td>LT27:295</td>
			<td>Mass: 175</td>
		</tr>
		<tr>
			<td>IFN&#947;</td>
			<td>Biolegend</td>
			<td>4S.B3</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>PTEN</td>
			<td>BD</td>
			<td>A2B1</td>
			<td>Mass: 159</td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>Biolegend</td>
			<td>Mab11</td>
			<td>Mass: 166</td>
		</tr>
		<tr>
			<td>Note: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling&#160; Kit (Fluidigm Cat: 201300) according to manufacturer protocol.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry

Las citoquinas juegan un papel fundamental en la patogénesis de enfermedades autoinmunes. Por lo tanto, la medición de los niveles de citocinas ha sido objeto de múltiples estudios en un intento de comprender los mecanismos precisos que conducen a la ruptura de la autotolerancia y autoinmunidad posterior. Enfoques hasta el momento se han basado en el estudio de un aspecto específico del sistema inmune (un solo o pocos tipos celulares o citocinas) y no ofrecen una evaluación global de la enfermedad autoinmune complejo. Mientras que estudios de sueros de pacientes han producido penetraciones importantes en autoinmunidad, no proporcionan la fuente celular específica de las citocinas de desajuste detectadas. Aquí se describe un enfoque integral de unicelular para evaluar la producción de citoquinas en varios subconjuntos de células inmunitarias, dentro del contexto de "intrínseco" específico para cada paciente de plasma factores de circulación. Este enfoque permite la monitorización de la paciente específico phenotype inmune (marcadores de superficie) y función (citoquinas), ya sea en su natural "intrínseca patógenos" enfermedad estatal, o en la presencia de agentes terapéuticos (in vivo o ex vivo).

Figura 1 muestra el flujo de trabajo para la estimulación y procesamiento de las muestras de sangre periférica, incluyendo asignación de alícuotas de la muestra de sangre, momento de la incorporación de agentes de estimulación, proteína transporte inhibidor de Cóctel e incubación veces hasta la lisis de glóbulos rojos (gr) y la fijación. La elección de los agentes estimulantes dependerá de las vías de señalización y citoquinas que son objeto d...

Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Análisis unicelular de Immunophenotype y producción de citoquinas en sangre periférica por citometría de masas

Aquí describimos un enfoque proteómico unicelular para evaluar inmunológico fenotípico y funcional (inducción de citocinas intracelulares) alteraciones en las muestras de sangre periférica, analizadas mediante citometría de masas.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

This method can help answer questions in the autoimmunity field such as identifying cell frequency abnormalities and function differences such as production of key cytokines in the setting of autoimmune disease. The main advantage of this method is that it can capture a broad repertoire or different cell types and function differences from a easily obtainable peripheral blood sample. Generally, individuals new to this process may struggle with getting the timing of the blood processing steps right, the antibody panel design, the barcoding process itself, and analysis of high dimensional single cell data.This systems immunology approach is particularly suitable for autoimmune diseases like SLE where immune dysregulation is pervasive and evident in peripheral blood. Visual demonstration of this method is important to clarify the timing of steps and also to show how multiple samples can be barcoded and pooled prior to mass cytometry analysis. To begin this procedure, place the labeled round bottom polystyrene tubes of five different conditions for whole blood processing on a rack.Next, add one milliliter of 37 degree Celsius RPMI to each tube. Then, add one milliliter of whole blood to each tube and pipette up and down several times to mix thoroughly with RPMI. Subsequently, add 10 microliters of pre-diluted ruxolitinib to the tube labeled T six plus five R and mix thoroughly.Place the rack of tubes in the incubator at 37 degrees Celsius and begin timing the incubation. To process the T zero sample, pipette the entire content of the T zero tube into a labeled conical tube containing 20 milliliters of 37 degree Celsius lyse/fix buffer at working concentration. To optimize cell recovery, rinse the tube with lyse/fix buffer and mix by inverting the conical tube.Incubate the cells at 37 degrees Celsius for 15 minutes to allow for lysis and fixation. Then, centrifuge the cells at 500 times G for five minutes at room temperature. Afterward, decant the supernatant and re-suspend the cells in one milliliter of ice cold PBS to break up the pellet.Then, fill the conical tube to a 15 milliliter volume with PBS. Subsequently, centrifuge the cells at 500 times G for five minutes at room temperature. Re-suspend the cells in one milliliter of CSM to break up the pellet.Then, transfer the sample to a labeled microcentrifuge tube for antibody staining. Using an automated cell counter, count the cells with a 10 microliter sample. Next, centrifuge the cells at 500 times G for five minutes at room temperature.Afterward, aspirate the supernatant, leaving the pellet in about 60 microliters of residual. Keep this pellet on ice until the samples from other conditions have completed processing. At T equals 30 minutes, transfer the tube rack to the tissue culture hood.Add 10 microliters of R848 at 0.1 grams per microliter to the T six plus R848 tube and mix thoroughly. Then, add 10 microliters of LPS at 0.01 micrograms per microliter to the T six plus LPS tube and mix thoroughly. Afterward, add four microliters of protein transport inhibitor to the tubes labeled T six, T six plus five R, and T six plus LPS and return the samples to the incubator until T equals six hours.Mix the samples thoroughly with a P1000 pipette every two hours. At T equals two hours, add four microliters of protein transport inhibitor cocktail to the T six plus R848 tube. Mix all samples and return the rack to the incubator until T equals six hours.At T equals four hours, mix the samples with a P1000 pipette one more time. At T equals six hours, process T six, T six plus LPS, T six plus R848, and T six plus five R blood sample tubes as described for the T zero sample. Then, store the pellets in residual CSM volume at minus 80 degrees Celsius to process later.To barcode the lysed, fixed cells, thaw the samples from the minus 80 degrees Celsius storage slowly on ice. Dilute the 10X barcoding perm buffer with PBS by one to 10 and make enough buffer for three milliliters per sample. Fill one non-sterile trough with CSM and one with the one X barcoding perm buffer.Then, add one milliliter of ice cold CSM to the freshly thawed samples, mix thoroughly, and transfer to the respective pre-labeled polypropylene cluster tubes. Now, take a 10 microliter sample and count the cells using an automated cell counter. Normalize the cell counts in each cluster tube by removing the volume of excess cells.Then, centrifuge the cells at 600 times G for five minutes at room temperature. Re-suspend the cells in one milliliter of one X barcoding perm buffer with a multichannel pipette and centrifuge at 600 times G for five minutes at room temperature. Afterward, aspirate off the supernatant.Line up the cluster tubes on a rack in the same order as indicated on the barcode key so the sample matches with its barcode. Add 800 microliters of one X barcoding perm buffer by multichannel pipette to all samples in the cluster tubes without touching the cell pellet to reduce cell loss. Then, set aside the rack with the cluster tubes.Remove the 20-plex palladium barcoding kit tube strips from minus 20 degrees Celsius and thaw at room temperature. Add 100 microliters of one X barcoding perm buffer, mix thoroughly, and transfer 120 microliters of the re-suspended barcode mix into the corresponding cell samples in the cluster tubes. Mix thoroughly by multichannel pipette so that there is no cross-contamination between individually barcoded samples.Incubate cluster tubes for 30 minutes at room temperature to allow the barcodes to label the cells. After 30 minutes, centrifuge the samples at 600 times G for five minutes at room temperature. Aspirate the supernatant, then re-suspend them in one milliliter of CSM.Subsequently, centrifuge and re-suspend in CSM again. Afterward, centrifuge at 600 times G for five minutes at room temperature and aspirate the supernatant. With a single pipette and using the same tip, transfer all cell pellets in about 70 to 80 microliters of residual volume to one polystyrene tube.Do not eject the pipette tip. Set aside the single pipette with this tip. With a multichannel pipette and new tips, add 100 microliters of CSM to each original cluster tube to maximize cell recovery.Using the single pipette with the tip that was set aside, transfer all cell pellets in 100 microliters of residual volume to the same polystyrene tube. Add CSM to top off the polystyrene tube to about three milliliters. Count and record the cell number of the pooled barcode set.Then, centrifuge at 600 times G for five minutes at room temperature and aspirate the supernatant. Proceed to staining the barcoded samples on the same day. This schematic demonstrates the workflow for the stimulation and processing of the peripheral blood samples, including allocation of blood sample aliquots, timing of the addition of stimulation agents, protein transport inhibitor cocktail, and incubation times until the red blood cell lysis and fixation.The choice of stimulating agents will depend on the signaling and cytokine pathways that are targeted for assessment. Following fixation and RBC lysis, the individual lysed, fixed cell samples from a healthy donor were barcoded, labeled with 26 antibodies against surface markers, permeabilized, and stained with 14 antibodies against cytokines. The limited gating strategy representing the identification of CD14 high monocytes is shown here.From left to right, each 2D plot represented is a population subset from the parent population that is gated from the 2D plot immediately to the left. Using CD14 high monocytes as representative in eight immune cell subsets, a mass cytometry analysis was performed on peripheral blood samples from a healthy donor at time zero unstimulated and following stimulation with TLR agonist LPS and R848 and the T-cell activator PMA ionomycin. An example of the cytokine induction in CD14 high monocytes is shown and selected cytokines with specificity to the stimulating agent used are depicted.R848 selectively induces IL-12p40 subunit and MCP1 in CD14 high monocytes while LPS does not. In contrast, PMA ionomycin does not induce cytokines in CD14 high monocytes. Once mastered, the blood stimulations can be done in about seven hours and the barcoding step can get done in just about an hour.When attempting this procedure, it's important to be mindful of the timing of the steps and to plan ahead accordingly. Once you have this procedure in place, you can consider altering the blood stimulation conditions in the mass cytometry panel in order to assess the immune cell responses that are specific to your own research aims. After watching this video, you should have a good understanding of how to elicit cytokine responses from whole blood samples and how to pool multiple samples into a barcoded set for mass cytometry analysis.

Research

JoVE Journal

Immunology and Infection

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Investigación

Inmunología e Infección

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

La enumeración de las principales poblaciones de leucocitos de sangre periférica en ensayos clínicos multicéntricos utilizando un ensayo de sangre entera Fenotipado

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

La disección de señalización inmune innata en Evasion Viral de la producción de citoquinas

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

La detección de glucógeno en las células mononucleares de sangre periférica con ácido periódico de Schiff tinción

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Generación de células T humanas aloantígeno-específicos de la sangre periférica

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Un enfoque semi-automatizado de preparación de cócteles de anticuerpos para el Análisis Inmunofenotípicos de la sangre periférica humana

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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

El aislamiento y la citometría de flujo análisis de las células mononucleares de sangre periférica Glioma infiltrantes

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

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

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 ...

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Perla base ensayo Multiplex para el análisis de perfiles de Cytokine de lágrima

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

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