Gli autori desiderano ringraziare Bing Zhang per la sua assistenza tecnica. Questo lavoro è stato finanziato dal Canadian Institutes for Health Research (MOP-86595). TAB è un investigatore CIHR Nuovo e Scholar AHFMR. QH è sostenuto da una borsa di studio CIHR Canada Graduate - Dottorato e un AIHS tempo pieno Studentship. SAN è sostenuto da una borsa di studio Queen Elizabeth II Graduate. AYWS è sostenuto da una borsa di studio post-laurea NSERC - Dottorato.

Fare riferimento alla Figura 1 per uno schema generale del protocollo sperimentale. 1. Dissezione <ol><li> Luogo sterile setaccio a maglia d&#39;acciaio in 60 x 15 millimetri capsula di Petri. Una unità è necessaria per campione di tessuto. </li><li> Aggiungere 5 ml di soluzione salina bilanciata di Hank (HBSS) a ogni piatto. Tenere i piatti sul ghiaccio. </li><li> Eutanasia topi con CO 2. </li><li> Del mouse sulla superficie protetta dissezione, lato ventrale rivolto verso l&#39;alto. Mouse spruzzo con 70% etanolo per la sterilizzazione e per assicurare che il pelo è arruffati giù. </li><li> Usando forbici chirurgiche, cominciano dissezione facendo una incisione nella pelle ventrale addominale appena sopra i genitali. Estendere incisione verso l&#39;alto fino al mento. </li><li> Dalla linea mediana, estendere l&#39;incisione giù lungo tutti gli arti. Tirare indietro la pelle flaccida e il pin verso il basso sulla superficie dissezione. </li><li> Raccolto il timo: <ol><li> Sollevare la punta inferiore dello sterno per fare un&#39;incisione.</li><li> Evitare il fegato, tagliare il diaframma per staccare la gabbia toracica e poi tagliare la gabbia toracica per ogni lato. Tagliare i cassa toracica verso l&#39;alto su entrambi i lati, avendo cura di evitare i polmoni e il cuore. </li><li> Estrarre delicatamente il retro cassa toracica con una pinza. Il timo è un bianco, organo bilobato situato sopra il cuore. Utilizzando il bordo piatto della pinza per afferrare la parte inferiore dei lobi, tirare delicatamente il timo e posizionarlo sullo schermo maglia. </li></ol></li><li> Raccogliere la milza: <ol><li> La milza è un rosso, a forma di tavola da surf organo situato sul lato sinistro della cavità addominale del mouse, sotto il fegato. </li><li> Effettuare un&#39;incisione nella cavità addominale. Estrarre delicatamente la milza con un paio di pinze, con la seconda coppia per prendere in giro via del tessuto connettivo. Posizionare la milza su uno schermo a rete separata. </li></ol></li></ol> 2. Preparazione cellulare <ol><li> Utilizzando uno stantuffo di una siringa da 3 ml, macinareorgani nello schermo maglia fino tessuto connettivo e solo resti di grasso. Sciacquare maglia con HBSS dal piatto tre volte. Utilizzare un pistone nuovo per ogni campione di tessuto. <ol><li> Altre opzioni per omogeneizzare tessuto può essere utilizzato al posto di questo metodo. </li></ol></li><li> Contare le cellule con un emocitometro. </li><li> Pellet di cellule per centrifugazione a 335 xg per 5 minuti a 4 ° C. </li><li> Splenociti Risospendere in 500 pl di tampone di lisi ACK (0,15 M NH 4 Cl, 10 mM KHC0 3, 0,1 mM Na 2 EDTA, pH 7,2) per 10 min a temperatura ambiente. Timociti Risospendere a 20 x 10 6 cellule / ml in FACS tampone (PBS, 1% FCS, sodio azide 0,02%) e mettere da parte su ghiaccio. </li><li> Ritorno splenociti di isotonicità aggiungendo 5 ml di HBSS. Splenociti pellet per centrifugazione e risospendere a 20 x 10 6 cellule / ml in tampone FACS. Se le cellule hanno bisogno di rimanere sterile, è possibile utilizzare sterile RPMI + 10% FCS. </li></ol> 3.Le cellule colorazione per Citometria a Flusso Lo scopo di questo protocollo è quello di delineare strategie per l&#39;analisi di selezione positiva e negativa utilizzando tipo selvatico (WT) e topi CD4 HY. Per considerazioni di carattere generale in materia di citometria a flusso di progettazione sperimentale, l&#39;acquisizione e l&#39;analisi, si rimanda ad un eccellente recensione di Tung et al 11. <ol><li> Aliquotare 4 x 10 6 timociti per campione di citometria a flusso per ciascun pozzetto di una piastra a 96 pozzetti, così come 1 x 10 6 splenociti WT per il controllo di compensazione. Se si utilizza topi transgenici, è necessario includere un topo WT come un controllo in un esperimento. </li><li> Bloccare i recettori Fc incubando le cellule con anti-CD16/32 (clone 2.4G2) per 10 min in ghiaccio. </li><li> Spin piastra a 335 xg a 4 ° C per 5 min. Togliere il coperchio e dissipare liquido dai pozzetti muovendo la piastra una volta, a faccia in giù in un lavandino. Risospendere ogni pozzetto in 200 pl di tampone FACS. Ripetere il lavaggio. </li><li> Preparare cocktail di anticorpi come di seguito elencati, usando la concentrazione ottimale di ciascun anticorpo base diluizione in 200 pl di tampone FACS per pozzetto. La concentrazione ottimale di ciascun anticorpo è definita come la concentrazione minima necessaria per fornire il più grande separazione di popolazioni positive e negative. Se non c&#39;è popolazione negativa per un determinato anticorpo, un anticorpo isotipo dovrebbero essere inclusi. Il volume totale per pozzetto può essere ridotta (ad esempio a 100 pl per pozzetto), se desiderato. <ol><li> WT timo: anti-TCRβ, anti-CD4, anti-CD8, anti-CD69 o anti-CD5, anti-CD24 </li><li> CD4 HY timo: anti-HY TCR (T3.70), anti-CD4, anti-CD8, anti-CD69 o anti-CD5, anti-CD24 Per ottenere una migliore separazione di CD69 e CD69 lo popolazioni hi in timociti, si raccomanda che un anticorpo biotinilato anti-CD69 primaria essere utilizzato, seguito da una macchia secondaria contenente fluorocromo coniugato streptavidina. Abbiamo use la stessa strategia per CD5 colorazione. </li></ol></li><li> Incubare le cellule con 200 ml di cocktail di anticorpi in tampone FACS per 30 min in ghiaccio, al buio. </li><li> In concomitanza con colorazione anticorpo cocktail, macchia ogni controllo di compensazione con un solo fluorocromo coniugato anticorpo. Idealmente, colorazione compensazione comporta gli stessi anticorpi utilizzati nel cocktail di anticorpi. Tuttavia, colorazione per ciascun antigene specifico non può essere fattibile per grandi esperimenti quando molti antigeni sono stati dosati. Pertanto, sia per colorazione anti-CD4 e anti-CD8 è raccomandato per l&#39;alta espressione di questi antigeni, o l&#39;uso di perline di compensazione. Macchia in tampone FACS per 30 min in ghiaccio, al buio. Lascia un controllo di compensazione senza macchia. </li><li> Lavare le cellule due volte con tampone FACS. </li><li> Risospendere le cellule in tampone FACS e trasferimento tubi FACS. </li><li> Acquisire campioni su citofluorimetro. Include l&#39;acquisizione di FSC-A e FSC-W a unallow di discriminazione doppietto. </li></ol> 4. Analisi dei dati Citometria a flusso - non TCR topi transgenici Usiamo FlowJo per citometria a flusso l&#39;analisi dei dati. Fare riferimento alla Figura 2A per la strategia di gating per i non-TCR topi transgenici. <ol><li> Utilizzo di FSC-A da SSC, cancello elettronico sulla popolazione &quot;linfociti&quot;. </li><li> All&#39;interno della popolazione &quot;linfociti&quot;, utilizzare FSC-A da FSC-W per via elettronica porta del FSC-W popolazione lo escludere doppietti cellulari e aggregrates. Questa è la porta &quot;singoletto&quot;. </li><li> Con gli eventi del cancello &quot;singoletto&quot;, la trama da CD4 CD8. Disegna cancelli per DN, DP sordo, DP luminoso, CD4 + CD8 popolazioni ecco, CD4SP e CD8SP come illustrato nella figura 2B. </li><li> Sotto le porte CD4SP e CD8SP, creare TCRβ da CD24 trame. Utilizzare lo strumento gating quadrante per disegnare cancelli come illustrato in Figura 2C. </li><li> Creare una new trama dal cancello &quot;singoletto&quot;: TCRβ da CD69. Disegnare porte A, B, C, D, come illustrato nella Figura 2D. </li><li> Creare un altro grafico dal cancello &quot;singoletto&quot;: TCRβ da CD5. Disegnare porte I, II, III, IV come illustrato nella Figura 2E. Questa è un&#39;altra strategia per l&#39;esame di selezione positiva. </li><li> Applicare DN, DP sordo, DP luminoso, int CD4, CD4SP e CD8SP porte da sotto &quot;singoletto&quot; ai cancelli i, ii, iii, iv, A, B, C, D (Figura 2D, E). </li><li> Calcolare il numero di cellule in un particolare sottoinsieme moltiplicando cellularità organo per la frequenza di ogni porta successiva fino a raggiungere la popolazione target. Per esempio, il numero di TCRβ hi-CD69 CD8SP timociti sarebbe determinata dal timo cellularità x% TCR BHI-CD69 (frazione D) x CD8SP%. <ol><li> Conteggio tiene già conto delle cellule vive e morte in modo da non includere il &quot;lymphocYTE &quot;porta nei calcoli. </li></ol></li></ol> 5. Analisi dei dati Citometria a flusso - TCR topi transgenici Fare riferimento alla Figura 3A per la strategia di gating per TCR topi transgenici. <ol><li> Seguire i punti 4.1 e 4.2, come nella sezione precedente. </li><li> Sotto il cancello &quot;singoletto&quot;, creare un T3.70 dalla trama SSC e cancello sul T3.70 + popolazione cellulare per analizzare antigene-specifiche cellule T (Figura 3B). In alcune circostanze, può essere interessante confrontare questa popolazione alla non-antigene specifico (T3.70) popolazione delle cellule T. </li><li> All&#39;interno della popolazione T3.70 +, disegnare DN, DP, CD4SP e CD8SP porte (Figura 3C). <ol><li> Per i campioni di controllo WT, disegnare questi cancelli sotto la porta &quot;singoletto&quot; o creare un T3.70-gate come ci saranno pochissimi T3.70 + cellule. </li></ol></li><li> Sotto il cancello CD8SP, creare unT3.70 da CD24 trama. Utilizzare lo strumento di gating quadrante per dividere le celle in quattro popolazioni (Figura 3F). </li><li> Creare un istogramma sovrapposto raffigurante CD69 espressione nel vano DP di topi WT e T3.70 + vano DP di CD4 HY topi (Figura 4A). Ripetere esaminare CD5 espressione (Figura 4B). </li><li> Calcolare il numero assoluto di cellule in ciascun sottoinsieme di interesse come in 4,8. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

Il protocollo qui presentato può essere utilizzato per esaminare selezione positiva e negativa in non-transgenici TCR e TCR topi transgenici. Questo protocollo descrive la colorazione degli antigeni di superficie. Per un&#39;ulteriore analisi dei meccanismi molecolari, è spesso necessario eseguire la colorazione intracellulare. Usiamo il BD Biosciences Cytofix / Cytoperm Kit per la maggior parte delle proteine ​​intracellulari e la Foxp3 BD Biosciences Kit di colorazione per fattori di trascrizione. Noi di solito ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Sale HyClone Hank soluzione equilibrata </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Rete metallica schermi </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Piastre di Petri (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Siringhe (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Provette coniche (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscopio </td><td> Zeiss - Primo Star </td><td> 415500-00XX-000 </td><td></td></tr><tr><td> Emocitometro </td><td> HausserScientifico </td><td> 3110 </td><td></td></tr><tr><td> Piastra a 96 pozzetti </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Pipetta multicanale </td><td> FISHERBRAND </td><td> 21-377-829 </td><td></td></tr><tr><td> Siero fetale di vitello </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Tampone fosfato salino </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> Sodio azide </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> FcR reagente bloccante </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-mouse HY TCR </td><td> eBioscience </td><td> XX-9930-AA * </td><td> Clone T3.70 </td></tr><tr><td> Anti-topo CD4 </td><td> eBioscience </td><td> XX-0042-AA * </td><td> Clone RM4-5 </td></tr><tr><td> Lanti-mouse CD8α </td><td> eBioscience </td><td> XX-0081-AA * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-topo CD24 </td><td> eBioscience </td><td> XX-0242-AA * </td><td> Clone M1/69 </td></tr><tr><td> Anti-mouse TCRβ </td><td> eBioscience </td><td> XX-5961-AA * </td><td> Clone H57-597 </td></tr><tr><td> Anti-topo CD69 Biotinylated </td><td> eBioscience </td><td> 13-0691-AA * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-mouse CD5 Biotinylated </td><td> eBioscience </td><td> 13-0051-AA * </td><td> Clone 53-7,3 </td></tr><tr><td> Streptavidina </td><td> eBioscience </td><td> XX-4217-AA * </td><td></td></tr><tr><td> Citometro di flusso </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tubi </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Citometria di flusso software di analisi </td><td> TreeStar - Flowjo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * Varia da XX e YY fluorocromo varia per grandezza flaconcino. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Un sistema immunitario sano è necessario che le cellule T rispondono agli antigeni estranei, pur rimanendo tolleranza di auto-antigeni. Riarrangiamento casuale del recettore delle cellule T (TCR) α e β loci genera un repertorio T cella con grande diversità nella specificità antigenica, sia per sé ed esteri. Selezione del repertorio durante lo sviluppo nel timo è critica per generare le cellule T sicuro e utile. Difetti di selezione timica contribuire allo sviluppo di malattie autoimmuni e immunodeficienza 1-4. Progenitori delle cellule T inserire il timo come doppi negativi (DN) timociti che non esprimono CD4 o CD8 co-recettori. Espressione del αβTCR ed entrambi co-recettori avviene a doppio positivo (DP) stadio. Interazione del αβTCR con auto-peptide-MHC (pMHC) presentata da parte delle cellule del timo determina il destino del timociti DP. Interazioni ad alta affinità portare alla selezione negativa e di eliminazionezione di auto-reattivi timociti. Bassa affinità risultato interazioni in selezione positiva e sviluppo di CD4 o CD8 singoli positivi (SP) cellule T capaci di riconoscere antigeni estranei presentati da auto-MHC 5. Selezione positiva può essere studiata in topi con un anticorpo (tipo selvatico) repertorio TCR osservando la generazione di cellule T mature. Tuttavia, essi non sono ideali per lo studio di selezione negativa, che comporta l&#39;eliminazione di piccoli antigene-specifiche popolazioni. Molti sistemi modello sono stati utilizzati per studiare selezione negativa, ma variano nella loro capacità di ricapitolare eventi fisiologici 6. Per esempio, stimolazione in vitro dei timociti manca dell&#39;ambiente timica che è intimamente coinvolto nella selezione, mentre la somministrazione di antigene esogeno può portare a non-specifico delezione di timociti 7-9. Attualmente, i migliori strumenti per lo studio in vivo selezione negativa sono topi che esprimono una transgENIC specifico TCR per endogena auto-antigene. Tuttavia, molti classici modelli transgenici TCR sono caratterizzati dalla prematura espressione della catena transgenico TCRα nella fase DN, con conseguente prematuro selezione negativa. Il nostro laboratorio ha sviluppato il modello HY cd4, in cui il transgenico HY TCRα condizionatamente espressa nella fase DP, consentendo la selezione negativa a verificarsi durante il DP di transizione SP come avviene nei topi di tipo selvatico 10. Qui, si descrive un protocollo basato citometria di flusso per esaminare timica selezione positiva e negativa nel topo HY modello CD4. Mentre la selezione negativa in topi CD4 HY è altamente fisiologica, questi metodi possono essere applicati anche ad altri modelli transgenici TCR. Ci sarà anche la presentazione delle strategie generali per l&#39;analisi di selezione positiva in un repertorio policlonale applicabile a tutti i topi geneticamente manipolati.

In fisiologiche TCR modelli transgenici e topi WT, selezione positiva inizia nella fase DP luminoso prima di passare alla fase di DP opaca dopo l&#39;incontro di antigene. Timociti DP opaco quindi inserire una transizione CD4 + CD8 lo stadio prima di diventare CD4SP o timociti CD8SP (Figura 2B). Timociti maturi SP sono caratterizzati da elevata espressione TCR e perdita di CD24 (Figura 2C). Mentre il CD8 da CD4 profilo può rivelare...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Vi presentiamo un metodo basato citometria di flusso per esaminare lo sviluppo delle cellule T<em&gt; In vivo</em&gt; Utilizzando topi geneticamente manipolati su un tipo selvatico o T sfondo recettore delle cellule transgeniche.

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 ...

examination-thymic-positive-negative-selection-flow

Research

JoVE Journal

Immunology and Infection

21.7K Views.  University of Alberta. Vi presentiamo un metodo basato citometria di flusso per esaminare lo sviluppo delle cellule T In vivo Utilizzando topi geneticamente manipolati su un tipo selvatico o T sfondo recettore delle cellule transgeniche.

Video: Examination of Thymic Positive and Negative Selection 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

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 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Lo studio delle interazioni di<em&gt; Staphylococcus aureus</em&gt; Con neutrofili mediante citometria a flusso e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Rilevamento di nanoparticelle fluorescenti Interazioni con Primaria immunitario sottopopolazioni cellulari mediante citometria di flusso

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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.

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

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of 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 ...

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 ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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. ...

Esame di Selezione timica positivi e negativi per Citometria a Flusso

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Misurazione dell'attività Calpain nelle celle fissi e Living in citometria a flusso

Umano In Vitro come strumento di screening per il riconoscimento di uno Stato precoce di squilibrio immunitario

Lo studio delle interazioni di