Riconosciamo il finanziamento il sostegno del Consiglio Nazionale Salute e Ricerca Medica (concedere APP605524, 490.037 e 1.047.082), la Australian Research Council (concedere DP12010061), la strategia di National Infrastructure Research Collaborative d&#39;Australia e il fondo di investimento Education del Dipartimento di Innovazione, Industria , Scienza e la ricerca. PML è un destinatario di un premio post-laurea australiano.

Tutte le procedure sono state condotte in conformità con le politiche di Macquarie University e conformi al Consiglio nazionale della sanità e ricerca medica (NHMRC) Codice australiano di pratica. Il lavoro è stato svolto sotto gli Etica accordo No ARA 2012/017 approvate e ottenuto da parte del Comitato Etico degli animali presso Macquarie University. Tutti gli esperimenti sono stati eseguiti su SJL / J topi se non indicato diversamente. 1. Mouse e Sperimentale Malaria Infezione <ol><li> Mice House a temperatura controllata (21 ° C), con un ciclo luce-buio 12:12 h. </li><li> Scongelare una aliquota di crioconservati P. chabaudi adami DS con 5% parassitati globuli rossi (pRBCs) e iniettare 200 ml nella cavità intraperitoneale di C57BL / 6 del mouse donatore. </li><li> Monitorare donatore del mouse parassitemia ogni 1 - 2 giorni in citometria a flusso, come descritto nella Sezione 3 - 4 del presente protocollo. Una volta che i donatori raggiunge 5-15% parassitemia, raccogliere il sangue dalla puntura cardiaca come segue: <ol><li>Aspirare 100 ml di soluzione di eparina (300 U / ml di eparina in soluzione di Ringer topo (MTR) (154 mM NaCl, KCl 5,6 mM, 1 mM MgCl 2, 2.2 mM CaCl 2, 20 mM HEPES, glucosio 10 mM, 30 U / ml eparina, pH 7,4, 0,22 mM filtro sterilizzato)) in una siringa da 1 ml con un ago 26 G. </li><li> Anestetizzare il mouse per inalazione con il 5% isofluorane, confermando la profondità desiderata di anestesia per mancanza di risposta ritiro del pedale e riflessi corneali. </li><li> Posizionare il mouse sul suo lato e perpendicolarmente ago inserto appena sotto il gomito, attraverso le costole, e nel cuore. Estrarre lo stantuffo della siringa lentamente e ruotare ago fino a 0,5 - si ottiene 1 ml di sangue. </li><li> Eseguire l&#39;eutanasia umana da dislocazione cervicale sotto anestesia. </li></ol></li><li> Calcolare i pRBCs per volume di sangue dei donatori assumendo un emocromo di 9 x 10 6 RBC / ml (ossia, se il donatore era al 10% parassitemia quando sacrificati, il sangue conterrà 9 x 10 5pRBC / ml). Diluire il sangue parassitati in Krebs tamponata salina (126 mM NaCl, 2,5 mM KCl, 25 mM NaHCO 3, 1.2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2.5 mM CaCl 2, 0.2% di glucosio, pH 7,2, 0,22 mM filtro sterilizzato) in modo che la concentrazione è 1 x 10 4 pRBCs per 200 microlitri e iniettare 200 microlitri nella cavità intraperitoneale di topi necessari per l&#39;etichettatura RBC e sperimentare trasfusione. </li><li> Monitorare parassitemia ogni 1 - 2 giorni in citometria a flusso, come descritto nei paragrafi 3-4 di questo protocollo. </li></ol> 2. Etichettatura dei globuli rossi e Transfusion <ol><li> Prelevare il sangue eparinizzato da topi mediante puntura cardiaca come descritto al punto 1.3, e disporli sul ghiaccio. Mantenere il sangue a 4 ° C in ogni momento. Raccogliere circa 200 ml di sangue per il mouse da iniettare. Se necessario, dividere il sangue in due campioni e trattare un campione, se lo desideri. NOTA: In questo modo, invasione del samp trattatale può essere paragonato ad un controllo, campione non trattato (vedere Figura 1 per schematica). </li><li> Preparare una soluzione 2x di ogni etichetta RBC fluorescente. <ol><li> Diluire a / ml soluzione madre 2 mg di Atto 633-N-Hydroxysuccinimide (Atto 633-NHS) in MTR modo che la concentrazione è di 20 mg / ml. </li><li> Diluire a / ml soluzione madre 25 mg di biotina-N-Hydroxysuccinimide (biotina-NHS) in MTR modo che la concentrazione di 250 ug / ml. </li></ol></li><li> Split campioni di sangue in due provette e aggiungere un volume uguale di soluzione di 2x Atto 633-NHS ad un tubo, e un volume uguale del 2x biotina-NHS per l&#39;altro tubo. Mescolare immediatamente. Incubare sangue a 4 ° C per 1 ora con costante miscelazione lenta, o in alternativa, miscelare campione ogni 10 - 15 minuti. </li><li> Lavare etichettati sangue 3 volte con MTRC (MTR + 0,5% di siero albumina bovina (BSA), 0,22 mM filtro sterilizzato) come segue: <ol><li> Aggiungere almeno 2 volumi di MTRC, centrifugare a 750 xg per 5 min, rimuovere il supernatante e resuspend il pellet in almeno 2 volumi di MTRC. Ripetere 2 volte. </li><li> Dopo l&#39;ultimo lavaggio, centrifugare a 750 xg per 5 minuti e combinare il sangue pellet in diverse combinazioni (ad esempio, non trattata Atto 633 / trattati biotina, non trattati biotina / trattata Atto 633). Aggiungere MTR per portare a un volume di 200 ml per topo da iniettare. </li></ol></li><li> Iniettare sangue marcato per via intravascolare in roditori malaria topi infettati o controlli non infetti come segue: <ol><li> Eseguire trasfusione a 2 - 15% parassitemia al culmine di parassita schizogonia. NOTA: ciò avviene circa a metà del ciclo di buio (cioè tra 12 - 2 am il normale ciclo di luce, o 12 - 2 pm un ciclo di luce inversa). </li><li> Topi calda usando una lampada di calore per 5-10 minuti per aumentare il flusso di sangue, ma di prendere precauzioni per non surriscaldare i topi. Inserire topi in un dispositivo di ritegno e iniettare per via intravascolare 200 microlitri di sangue marcato (circa 1 - 2 x 10 9 RBCs) attraverso la vena della coda. </li></ol></li><li> Raccogliere campioni di sangue in diversi momenti dopo l&#39;iniezione, come descritto nella Sezione 3. NOTA: i tassi di invasione possono essere quantificabili in meno di 10 minuti dopo l&#39;iniezione, a seconda parassitemia del mouse destinatario. </li></ol> 3. Raccolta di campioni di sangue e di preparazione per Citometria a Flusso <ol><li> Preparare 50 ml di soluzione colorante per campione, più extra, il giorno dell&#39;esperimento come segue: <ol><li> Scongelare una aliquota di 6 JC-1 mM conservati a -20 ° C in dimetil solfossido (DMSO). </li><li> Caldo 50 ml di MTRC per campione, più extra, a 37 ° C. </li><li> Mentre continua il vortex MTRC pre-riscaldato, aggiungere JC-1 in modo che la concentrazione finale è di 12 micron. Questo viene fatto per ridurre la formazione di aggregati JC-1; se aggregati si formano, fare tubo non centrifuga in modo da evitare la colorazione. NOTA: Una piccola quantità di aggregazione non influirà risultati. </li><li> Aggiungi anti-CD45 APC eFluor 780 e anti-CD71 PerCP eFluor 710 in modo che la concentrazione finale è di 1 mg / ml. Se si esegue l&#39;esperimento RBC etichetta, aggiungere streptavidina PE-Cy7 in modo che la concentrazione finale è di 1 mg / ml. </li></ol></li><li> Eseguire sanguinamento coda da amputazione della punta della coda o lacerazione del vaso sanguigno nella coda. Mettere una goccia di sangue di coda su una piccola barca o pesare vetrino. Dopo la raccolta del sangue, assicurarsi che l&#39;emorragia si è fermata. Pipettare 3 ml di sangue coda in 50 ml di soluzione colorante pre-riscaldato a 37 ° C e incubare i campioni a 37 ° C per 20 min. </li><li> Scongelare una aliquota di 4 mM Hoechst 33342 conservati a -20 ° C in acqua distillata (se si utilizza un laser 355 nm) o 2 mM Hoechst 34580 conservati a -20 ° C in acqua distillata (se si utilizza un laser 405 nm). Preparare 500 microlitri per campione, più extra, di una soluzione di 4 micron o 2 micron di Hoechst in MTRC rispettivamente. </li><li> Aggiungere 500 ml di 4 micron Hoechst33342 o 2 mM Hoechst 34580 campioni e incubare a temperatura ambiente per 20 min. </li><li> Cellule centrifugare a 750 xg per 3 min a 4 ° C, scarto surnatante, aggiungere 700 microlitri MTRC e analizzare mediante citometria a flusso. </li></ol> 4. Citometria a flusso <ol><li> Analizzare i campioni utilizzando un laser 355/488/633 nm o 405/488/633 strumento laser nm. </li><li> Filtrare i campioni attraverso un colino cella di 35 micron immediatamente prima dell&#39;analisi. Risciacquare filtro cella con acqua distillata tra campioni e riutilizzo. </li><li> Registra abbastanza eventi in modo che la popolazione più piccola contiene almeno 500 eventi (di solito 1.000.000 - 10.000.000 eventi totali per campione). </li><li> Excite Hoechst 33342 utilizzando un laser 355 nm e rilevare eventi attraverso un filtro 460/50. Excite Hoechst 34580 utilizzando un laser 405 nm e rilevare gli eventi attraverso un filtro 460/50. Excite JC-1, anti-CD71 PerCP eFluor 710 e PE-Streptavidina Cy7 utilizzando un laser 488 nm e rilevare gli eventi attraverso una 530/40, 692/40, e 750LP filtrare rispettivoly. Excite Atto 633 e anti-CD45 APC eFluor 780 utilizzando un laser 633 nm e di rilevare, rispettivamente, gli eventi attraverso un filtro 670/30 o 750LP. </li><li> Eseguire l&#39;analisi e la compensazione con flusso adeguato citometria software di analisi come segue: <ol><li> Selezionare cellule intere ed escludere rumore, detriti e piastrine basato su FSC / proprietà SSC (Figura 2A). Selezionare singole cellule basate su entrambi larghezza di impulso di trigger (Figura 2B) o utilizzando l&#39;area del picco FSC al rapporto di altezza (Figura 2C). </li><li> Selezionare globuli rossi maturi, ed escludere progenitori e leucociti RBC, sulla base di fluorescenza negativo nei PerCP eFluor 710 e APC eFluor 780 canali (Figura 2D). </li><li> Se si esegue l&#39;esperimento RBC etichetta, selezionare ogni popolazione di globuli rossi etichettati sulla base di PE-Cy7 e Atto 633 fluorescenza e analizzare questi separatamente (Figura 3A). </li><li> Selezionare pRBCs basata su fluorescenza positiva sia JC-1 e Hoechst ( <strong&gt; Figura 2E-H). </li></ol></li></ol> 5. Calcoli e Statistiche <ol><li> Calcolare parassitemia dividendo il numero di pRBCs per il numero totale dei globuli rossi (cioè, il numero di eventi in cancello Q2 e il numero di eventi in porta G4). Nell&#39;effettuare la parassitemia dosaggio invasione dei due popolazioni etichettati può essere calcolata dividendo il numero di pRBCs etichettate per il numero di globuli rossi etichettati in quella popolazione (cioè, il numero di eventi che sono sia porte L1 e Q2, diviso per il numero di eventi in porta L1). NOTA: etichetta parassitemia varia notevolmente a seconda di fattori quali la parassitemia endogena e il numero di merozoiti circolanti. Per questo motivo, è utile per segnalare il rapporto parassitemia, che viene calcolato come parassitemia della popolazione trattata etichettato diviso per il parassitemia della popolazione di controllo etichettata in ciascun topo. Per correggere eventuali effetti tintura, rapporto risulta uns un rapporto medio parassitemia tra i due combinazioni di tintura. </li><li> Determinare la significatività statistica dei rapporti con il t-test un campione con 1 come media ipotetica. </li></ol>

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Abbiamo descritto un metodo per la misura sia parassitemia e merozoite invasione campioni in vivo. In termini di misurazione parassitemia, questo metodo offre un vantaggio rispetto ai metodi precedenti 10-13 in quella HJ-globuli rossi possono essere distinti da pRBCs, riducendo così il numero di eventi positivi falsi. Mentre HJ-globuli rossi sono di solito rare negli esseri umani, alcuni studi riportano livelli elevati nei topi 15,16 la distinzione tra queste cellule e pRBCs importanti pe...

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.

Misura della parassitemia. Per la misura di parassitemia, cellule del sangue devono prima essere selezionati, e il rumore, detriti e piastrine esclusi, sulla base di FSC / SSC proprietà (Figura 2A). A seconda del citometro utilizzato, singole cellule devono poi essere selezionati sulla base sia di larghezza di impulso di trigger (Figura 2B), o altezza del picco FSC rapporto dell&#39;area (Figura 2C). Rimanendo eventi dovrebbero consistere di l...

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

Gli invade parassita della malaria e replicati all&#39;interno delle cellule rosse del sangue. La valutazione accurata di invasione merozoite e parassitemia è quindi fondamentale per la valutazione nel corso di infezione malarica. Qui si descrive un protocollo basato citometria a flusso per la misurazione di questi parametri in un modello murino di malaria.

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

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

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Research

JoVE Journal

Immunology and Infection

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

9.5K Views.  Macquarie University.  Gli invade parassita della malaria e replicati all'interno delle cellule rosse del sangue. La valutazione accurata di invasione merozoite e parassitemia è quindi fondamentale per la valutazione nel corso di infezione malarica. Qui si descrive un protocollo basato citometria a flusso per la misurazione di questi parametri in un modello murino di 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.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

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

Ricerca

Immunologia e infezioni

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.

La valutazione quantitativa delle cellule immunitarie nel tessuto danneggiato del midollo spinale mediante citometria di flusso: un nuovo uso per un metodo di purificazione cellulare

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.

Umano In Vitro</emSoppressione> come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario

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.

Esame di Selezione timica positivi e negativi per Citometria a Flusso

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.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

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 valutazione della retina microglia fagocitaria Funzione<em&gt; In Vivo</em&gt; Usando un test citometria a flusso basato su

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.

Flusso analisi di citometria di Particle-bound Bet v 1 allergene 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 ...

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.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

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).

Caratterizzazione dei sottoinsiemi di monociti umani da sangue intero flusso Cytometry analisi

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.

Discriminazione dei sette sottoinsiemi delle cellule immuni di due-fluorocromo flusso Cytometry

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