Reconocemos el apoyo financiero de la Salud y medicina Consejo Nacional de Investigación (subvención APP605524, 490.037 y 1.047.082), el Consejo Australiano de Investigación (subvención DP12010061), la Estrategia de Infraestructuras de Investigación Cooperativa Nacional de Australia y el fondo de inversión de Educación del Departamento de Innovación, Industria , Ciencia e Investigación. PML es un receptor de un premio de postgrado australiano.

Todos los procedimientos se realizaron de conformidad con las políticas de la Universidad de Macquarie y conformados a la Salud y medicina Consejo Superior de Investigaciones Científicas (NHMRC) Código australiano de la práctica. El trabajo se realizó en las Ética acuerdo No ARA 2012/017 aprobado y obtuvo del Comité de Ética Animal de la Universidad de Macquarie. Todos los experimentos se realizaron en ratones SJL / J menos que se indique lo contrario. 1. Los ratones y Experimental Malaria infección <ol><li> Los ratones bajo temperatura controlada (21 ° C) con un ciclo de luz-oscuridad de 12:12 h. </li><li> Descongelar una alícuota de criopreservados P. DS adami chabaudi con un 5% en los glóbulos rojos parasitados (pRBCs) y inyectar 200 l en la cavidad intraperitoneal de C57BL / 6 donantes ratón. </li><li> Monitorear donante parasitemia ratón cada 1 - 2 días por citometría de flujo como se describe en la Sección 3 - 4 de este protocolo. Una vez que los donantes alcanza 5-15% de parasitemia, recoger la sangre por punción cardiaca de la siguiente manera: <ol><li>Aspirar 100 l de solución de heparina (300 U / ml de heparina en solución Ringer ratón (MTR) (NaCl 154 mM, KCl 5,6 mM, 1 mM MgCl2, 2,2 mM CaCl 2, HEPES 20 mM, glucosa 10 mM, 30 U / ml heparina, pH 7,4, 0,22 mM de filtro esterilizado)) en una jeringa de 1 ml con una aguja de 26 G. </li><li> Anestesiar el ratón por inhalación con 5% de isofluorano, confirmando la profundidad deseada de la anestesia por la ausencia de respuesta de retirada de pedal y los reflejos corneales. </li><li> Coloque el ratón sobre su lado y la aguja de inserción perpendicularmente por debajo del codo, a través de las costillas, y en el corazón. Saque émbolo de la jeringa lentamente y girar la aguja hasta 0.5 - se obtiene 1 ml de sangre. </li><li> Realizar la eutanasia por dislocación cervical bajo anestesia. </li></ol></li><li> Calcular los pRBCs por volumen de sangre del donante asumiendo un recuento de sangre de 9 x 10 6 RBC / l (es decir, si el donante estaba en 10% de parasitemia cuando sacrificado, la sangre contiene 9 x 10 5GRP / l). Diluir la sangre parasitada en solución salina Krebs (NaCl 126 mM, KCl 2,5 mM, 25 mM de NaHCO3, 1,2 mM NaH 2 PO 4, 1.2 mM MgCl2, 2,5 mM CaCl 2, 0,2% de glucosa, pH 7,2, 0,22 mM de filtro esterilizado) tamponada de manera que la concentración es de 1 x 10 4 pRBCs por 200 l 200 l e inyectar en la cavidad intraperitoneal de ratones requeridos para el etiquetado RBC y el experimento transfusión. </li><li> Monitorear parasitemia cada 1 - 2 días por citometría de flujo como se describe en las secciones 3-4 de este protocolo. </li></ol> 2. El etiquetado de los glóbulos rojos y transfusión <ol><li> Recoger la sangre heparinizada a partir de ratones por punción cardiaca como se describe en el paso 1.3, y colocar en hielo. Mantener la sangre a 4 ° C en todo momento. Recoger aproximadamente 200 l de sangre por ratón a inyectar. Si es necesario, dividir la sangre en dos muestras y tratar una muestra lo deseas. NOTA: En este modo, la invasión de la samp tratadaLe puede ser comparado con un control, la muestra sin tratar (véase la Figura 1 para esquemática). </li><li> Preparar una solución 2x ​​de cada etiqueta RBC fluorescente. <ol><li> Se diluye una solución madre de 2 mg / ml de Atto 633-N-hidroxisuccinimida (Atto 633-NHS) en MTR modo que la concentración es 20 mg / ml. </li><li> Se diluye una solución madre de 25 mg / ml de biotina-N-hidroxisuccinimida (biotina-NHS) en MTR modo que la concentración es 250 mg / ml. </li></ol></li><li> Dividir las muestras de sangre en dos tubos y añadir un volumen igual de 2X solución Atto 633-NHS a un tubo, y un volumen igual de 2x la biotina-NHS al otro tubo. Mezclar inmediatamente. Incubar la sangre a 4 ° C durante 1 hora con mezclado lento constante, o alternativamente, la mezcla de la muestra cada 10 - 15 minutos a. </li><li> Lavar la etiqueta de sangre 3 veces con MTRC (MTR + 0,5% de albúmina de suero bovino (BSA), 0,22 mM de filtro esterilizado) como sigue: <ol><li> Añadir al menos 2 volúmenes de MTRC, centrífuga a 750 g durante 5 min, separar el sobrenadante, y resuspend el sedimento en al menos 2 volúmenes de MTRC. Repite 2 veces más. </li><li> Después del lavado final, se centrifuga a 750 xg durante 5 min y combinar la sangre se sedimentaron en diferentes combinaciones (es decir, sin tratar Atto 633 / biotina tratado, sin tratamiento de biotina / tratada Atto 633). Añadir MTR para hacer hasta un volumen de 200 l por ratón a inyectar. </li></ol></li><li> Inyectar la sangre marcada por vía intravascular en ratones infectados de malaria de roedores o controles no infectados como sigue: <ol><li> Realizar transfusión en 2-15% de parasitemia en el pico de la esquizogonia parásito. NOTA: esto ocurre aproximadamente a medio camino a través de la oscuridad ciclo (es decir, entre 12 a 2 de la mañana un ciclo de luz normal, o 12-2 pm en un ciclo de luz de marcha atrás). </li><li> Ratones calientes utilizando una lámpara de calor durante 5-10 minutos para aumentar el flujo sanguíneo, pero tomar precauciones de no sobrecalentar los ratones. Coloca los ratones en un dispositivo de contención y intravascular inyectar 200 l de sangre marcada (aproximadamente 1 - 2 x 10 9 RBCs) a través de la vena de la cola. </li></ol></li><li> Recoger muestras de sangre en diferentes momentos después de la inyección, como se describe en la Sección 3. NOTA: las tasas de invasión se pueden cuantificar menos de 10 min después de la inyección, dependiendo de parasitemia del ratón receptor. </li></ol> 3. Recolección de muestras de sangre y preparación para Citometría de Flujo <ol><li> Preparar 50 l de solución de tinción por muestra, y un poco más, en el día del experimento como sigue: <ol><li> Descongelar una alícuota de 6 mM JC-1 se almacena a -20 ° C en sulfóxido de dimetilo (DMSO). </li><li> Cálido 50 l de MTRC por muestra, y un poco más, a 37 ° C. </li><li> Mientras vórtice continuamente la MTRC pre-calentado, añadir JC-1, de modo que la concentración final es 12 mM. Esto se hace para reducir la formación de JC-1 agregados; si los agregados se forman, hacer tubo no centrífuga ya que esto evitará manchas. NOTA: Una pequeña cantidad de agregación no afectará a los resultados. </li><li> Añadir anti-CD45 APC eFluor 780 y anti-CD71 PerCP eFluor 710 de manera que la concentración final es 1 mg / ml. Si se realiza el experimento RBC marcado, añadir estreptavidina PE-Cy7 modo que la concentración final es 1 mg / ml. </li></ol></li><li> Realizar el sangrado de la cola por la amputación de la extremidad de la cola o laceración del vaso sanguíneo dentro de la cola. Coloque una gota de sangre de la cola a un pequeño sopesar barco o portaobjetos. Después de la extracción de sangre, asegúrese de que el sangrado se ha detenido. Pipetear 3 l de sangre de la cola en 50 l de solución de tinción pre-calentado a 37 ° C e incubar las muestras a 37 ° C durante 20 min. </li><li> Descongelar una alícuota de 4 mM Hoechst 33342 se almacena a -20 ° C en agua destilada (si se utiliza un láser de 355 nm) o 2 mM Hoechst 34580 se almacena a -20 ° C en agua destilada (si se utiliza un láser de 405 nm). Preparar 500 l por muestra, y un poco más, de una solución 4 M o 2 M de Hoechst en MTRC, respectivamente. </li><li> Añadir 500 l de 4 micras Hoechst33342 o 2 mM Hoechst 34580 a las muestras y se incuba a temperatura ambiente durante 20 min. </li><li> Centrifugar las células a 750 xg durante 3 min a 4 ° C, el sobrenadante de descarte, añadir 700 l MTRC y analizan por citometría de flujo. </li></ol> 4. Citometría de Flujo <ol><li> Analizar las muestras usando un láser 355/488/633 nm o 405/488/633 instrumento láser nm. </li><li> Filtrar las muestras a través de un filtro de células de 35 mM inmediatamente antes del análisis. Enjuague células colador con agua destilada entre muestras y reutilización. </li><li> Registre suficientes eventos para que la población más pequeña contiene al menos 500 eventos (generalmente 1000000 - 10000000 eventos totales por muestra). </li><li> Excite Hoechst 33342 utilizando un láser de 355 nm y detectar eventos a través de un filtro de 460/50. Excite Hoechst 34580 utilizando un láser de 405 nm y detectar eventos a través de un filtro de 460/50. Excite JC-1, anti-CD71 PerCP eFluor 710 y estreptavidina PE-Cy7 usando un láser de 488 nm y detectar eventos a través de un 530/40, 692/40, y 750LP filtro respectivaly. Excite Atto 633 y anti-CD45 de APC eFluor 780 usando un láser de 633 nm y detectar eventos a través de un filtro de 670/30 o 750LP respectivamente. </li><li> Realizar análisis y compensación mediante citometría de flujo apropiado de software de análisis de la siguiente manera: <ol><li> Seleccione células completas y excluir el ruido, los residuos y las plaquetas en base a FSC / SSC propiedades (Figura 2A). Seleccionar las células individuales sobre la base de cualquiera de ancho de pulso de disparo (Figura 2B) o utilizando el área del pico FSC a altura (Figura 2C). </li><li> Seleccione glóbulos rojos maduros, y excluir a los progenitores y los leucocitos RBC, basados ​​en fluorescencia negativa en los PerCP eFluor 710 y APC eFluor 780 canales (Figura 2D). </li><li> Si se realiza el experimento RBC marcado, seleccionar cada población de glóbulos rojos marcados basados ​​en PE-Cy7 y Atto 633 de fluorescencia y análisis de estos por separado (Figura 3A). </li><li> Seleccione pRBCs basado en fluorescencia positiva tanto para JC-1 y Hoechst ( <strong&gt; Figura 2E-H). </li></ol></li></ol> 5. Cálculos y Estadísticas <ol><li> Calcular parasitemia dividiendo el número de pRBCs por el número total de glóbulos rojos (es decir, número de eventos en Q2 puerta dividido por el número de eventos en G4 puerta). Al llevar a cabo la parasitemia ensayo de invasión de las dos poblaciones marcadas se puede calcular dividiendo el número de pRBCs marcadas por el número de glóbulos rojos marcados en esa población (es decir, el número de eventos que se encuentran en ambas puertas L1 y Q2, dividido por el número de eventos en puerta L1). NOTA: Etiquetado parasitemia varía considerablemente dependiendo de factores como la parasitemia endógeno y número de merozoitos circulantes. Por esta razón, es útil para informar de la relación de parasitemia, la cual se calcula como la parasitemia de la población marcada tratados dividido por la parasitemia de la población control con la etiqueta en cada ratón individual. Para corregir los posibles efectos de tinte, el informe de los resultados de uns una relación media de la parasitemia a través de las dos combinaciones de colorantes. </li><li> Determinar la significación estadística de los coeficientes utilizando la muestra de t-test con 1 como la media hipotética. </li></ol>

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Los autores no tienen nada que revelar.

Hemos descrito un método para la medición tanto de parasitemia y merozoite invasión de muestras in vivo. En términos de medición parasitemia, este método ofrece una ventaja sobre los métodos anteriores 10-13 en que HJ-glóbulos rojos pueden distinguirse de pRBCs, reduciendo así el número de eventos positivos falsos. Mientras HJ-glóbulos rojos son generalmente raros en los seres humanos, algunos estudios informan altos niveles en los ratones 15,16 hacer la distinción entre estas ...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Life Technologies</td>
			<td>T-3168</td>
			<td>Store small aliquots of 6mM stock solution at -20 &#176;C in DMSO</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-eFluor 780</td>
			<td>eBioscience</td>
			<td>47-0451-80</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD71 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0711-80</td>
			<td>Clone R17217</td>
		</tr>
		<tr>
			<td>Atto 633 NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>1464</td>
			<td>Atto 633-NHS. Store a 2mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>21335</td>
			<td>Biotin-NHS. Store a 25mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>Streptavidin PE-Cy7</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H478</td>
			<td></td>
		</tr>
		<tr>
			<td>35&#181;M filter cap tubes</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD FACSAria II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD Influx</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: CyAn ADP Analyzer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Medición de la parasitemia. Para la medición de la parasitemia, células de la sangre primero deben ser seleccionados, y el ruido, la suciedad y las plaquetas excluidos, basado en las propiedades del FSC / SSC (Figura 2A). Dependiendo del citómetro utilizado, las células individuales entonces deben seleccionarse en base a cada ancho de pulso de disparo (Figura 2B), o la altura del pico FSC relación de área (Figura 2C). Resto de eventos de...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Los invade parásito de la malaria y se replica dentro de las células rojas de la sangre. La evaluación precisa de la invasión merozoite y parasitemia es por lo tanto crucial para evaluar el curso de la infección por malaria. Aquí se describe un protocolo basado en citometría de flujo para la medición de estos parámetros en un modelo de ratón de la malaria.

En la Evaluación in vivo de roedores Plasmodium parasitemia y Merozoite Invasión por Citometría de Flujo

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.5K vistas.  Macquarie University.  Los invade parásito de la malaria y se replica dentro de las células rojas de la sangre. La evaluación precisa de la invasión merozoite y parasitemia es por lo tanto crucial para evaluar el curso de la infección por malaria. Aquí se describe un protocolo basado en citometría de flujo para la medición de estos parámetros en un modelo de ratón de la malaria. 

Video: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Evaluación cuantitativa de las células inmunes en el tejido de la médula espinal lesionada por citometría de flujo: un nuevo uso de un método de purificación celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

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

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

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 ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discriminación de siete subconjuntos de células inmunes por dos fluorocromo citometría de flujo

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...