Dr. Pramod Nehete e la signora Bharti Nehete macaco di sangue e campioni PBMC; Questo lavoro è stato parzialmente supportato con fondi dal NIH concedere AI 46969, e tutti i terreni di coltura cellulare sono stati prodotti dai Media Central Laboratory, che è finanziato dal NIH concedere CA 16672.

Le procedure per l&#39;analisi dettagliate fenotipica e funzionale del totale così come sottoinsiemi di cellule T di memoria può essere diviso in quattro parti: 1) Preparazione delle cellule e stimolazione antigenica, 2) indicatore colorazione superficiale, 3) colorazione delle citochine intracellulari e 4) La citometria a flusso analisi. 1. Preparazione delle cellule e la stimolazione antigenica <ol><li> Sia appena isolato e crio-conservato le cellule mononucleate del sangue periferico (PBMC) sono stati utilizzati in questo protocollo. Le PBMC sono state isolate da campioni venoso eparinizzato o citrato sangue dei macachi rhesus per sedimentazione gradiente di densità con Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO). Aliquote di PBMC sono stati congelati e conservati in 90% di FCS e 10% di DMSO in azoto liquido. </li><li> Quando si utilizza la crio-conservato PBMC, le fiale di surgelati PBMC sono stati rimossi da azoto liquido e rapidamente scongelato in un bagno d&#39;acqua 37 C, delicatamente mescolato, lavati con RPMI-1640 (Hyclone laboratori, Logan, UT) per rimuovere il supporto di congelamento e risospese in mezzi di comunicazione completo [CM; RPMI-1640 integrano con il 10% di siero FCS (Hyclone laboratori), 2mM L-glutamina (Sigma-Aldrich), 100U/ml penicillina / streptomicina (Invitrogen), e colto in 6 - piastre da coltura tissutale overnight a 37 ° C in un umidificata al 5% CO 2 nell&#39;atmosfera. La mattina seguente, conteggio delle cellule vitali sono stati determinati dal trypan blu metodo di esclusione del colorante e risospese in CM. </li><li> Le cellule sono state trattate in modo diverso per non specifici contro l&#39;antigene specifico per la stimolazione seguita da analisi di citochine: (a) Per i non-specifica stimolazione con forbolo miristato 12-13-acetato (PMA) e ionomicina (I) (Sigma-Aldrich, S. Louis, MO), aliquote di cellule in un volume di 0,1 ml (1,0 x 10 6 cellule / pozzetto) sono stati placcati in singoli pozzetti di piastre a 96 pozzetti tessuto cultura (BD Biosciences, Franklin lago, NJ). Il PMA e ionomicina sono stati utilizzati ad una concentrazione finale di 50ng/ml e 500ng/ml, rispettivamente. Il volume totale finale per l&#39;96-pozzetti coltura tissutale è 0.2ml/well: (b) Per la stimolazione antigene specifico, 1,0 x 10 6 cellule in un volume di 1,0 ml sono stati placcati in singoli pozzetti di piastre tessuto 24-ben cultura (BD Biosciences , Franklin lago, NJ), dove i pozzi sono stati pre-trattati come descritto in precedenza con modifica 9. In breve, il 24-piastre da coltura tissutale sono stati rivestiti con 2,5 mg / ml / pozzetto di capra anti-topo IgG (H + L) (Kierkegaard e Perry Laboratorio, Gaithersburg, MD) nella soluzione di 50 mM Tris (PH 8,6) per 4 ° C durante la notte. La mattina seguente sono stati lavati i piatti due volte con PBS sterile e co-stimolatorie mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) sono stati aggiunti a 10 mcg / ml / pozzetto seguito da incubazione per un&#39;ora a 37 ° C. Dopo l&#39;incubazione, le piastre sono state lavate due volte con PBS sterile a temperatura ambiente. Gli antigeni di interesse (tra cui cocktail di sei peptidi busta HIV 8 sono stati aggiunti ad una concentrazione finale di 10μg/ml di ogni peptide. Pozzi supplementari con le cellule in CM solo sono stati preparati come controllo negativo. Il volume totale finale per ciascun pozzetto delle 24 -pozzetti coltura tissutale era 1,0 ml. </li><li> Le cellule sono state coltivate per 6 ore a 37 ° C in un umidificata al 5% CO 2 nell&#39;atmosfera. Brefeldin A (Sigma-Aldrich, St. Louis, MO) è stato aggiunto alla cultura in 10μg/ml per la finale di 4,5 ore di stimolazione. Successivamente, le cellule sono state trasferite in provette da 5 ml in polipropilene e lavate con acqua fredda (4 ° C), tampone di lavaggio di flusso (Dulbecco PBS (DPBS, Ca 2 / Mg 2-libero; Life Technology, Rockville, MD) con 2% di siero FCS), e poi elaborati per la colorazione con i vari anticorpi fluorescenza etichettati. </li></ol> 2. Marcatori di superficie immunofluorescenza colorazione <ol><li> Cellule stimolate sono stati colorati con 1UL vivo / morto risolvibile acqua colorante fluorescente reattiva a 4 ° C al buio per 30 minuti, una volta lavate con tampone lavaggio freddo flusso. </li><li> Poi, i seguenti opportunamente titolato fluorescenza marcati anticorpi monoclonali sono stati aggiunti alle cellule: anti-CD3 PE-Cy7 (SP34-2), anti-CD8 Alexa700 (RPA-T8), anti-CD28-PerCP Cy5.5 (L293) , anti-CD95 APC (DX2) e anti CD4 Pacific Blue (OKT4), il tutto da BD Biosciences (Franklin lago, NJ), ad eccezione di CD4 blu pacific (OKT4) da eBioscience (San Diego, CA). Le cellule sono state incubate per 30 minuti a 4 ° C al buio. </li><li> Per ogni esperimento sia compensazione controlli e fluorescenza meno uno (FMO) 10,11 controlli sono stati utilizzati. Controlli di compensazione sono stati utilizzati per determinare la coeffecients spillover di ogni individuo macchia in altri rivelatori. Per i controlli di compensazione, un set completo di tubi contenenti sospensioni cellulari da uno di scimmia sono stati marcati con fluorescenti coniugati mAb singolarmente come macchie di colore. FMO è una combinazione di macchie multicolore che contiene tutti i reagenti, ma quello di interesse e viene utilizzato per determinare il confine tra un positivo unpopolazione ° negativo duplicando auto-fluorescenza livello e dati presenti nel campione completamente colorato. </li><li> Cellule colorate sono state poi lavate con tampone di lavaggio freddo flusso, e fissati in fissazione / permeabilizzazione soluzione (BD Biosciences, San Jose, CA) per almeno 10 minuti (in questa fase le cellule possono essere conservate durante la notte a 4 ° C al buio) prima di eseguire la colorazione intracellulare di citochine. </li></ol> 3. Citochine colorazione intracellulare <ol><li> Cellule fissate sono state lavate con tampone flusso di lavaggio, e poi Perm / Tampone di lavaggio (BD Biosciences, San Jose, CA) è stato aggiunto in base alle istruzioni del costruttore. In breve, le cellule sono state incubate in 0,1 ml di Perm 1X / Tampone di lavaggio per 10 minuti a 4 ° C al buio. </li><li> Successivamente, le cellule sono state incubate con il opportunamente titolato FITC anti-IFN-γ (B27) e PE-etichettati anti-IL-2 (MQ1-17H12) per 60 minuti a 4 ° C al buio. </li><li> Quindi le cellule sono state lavate due volte con la soluzione di permeabilizzazione </li><li> Dopo l&#39;ultimo lavaggio, le cellule sono state risospese in 1% paraformaldeide in DPBS e sottoposti ad analisi di citometria a flusso entro 24 ore. </li></ol> 4. Analisi di citometria a flusso <ol><li> Le celle colorate sono state acquisite su un citometro a flusso II LSR (BD Biosciences, San Jose, CA) o ciano ADP (Dako, Carpinteria, CA). FACS dati sono stati analizzati utilizzando il software FlowJo (Albero Star, Ashland, OR). </li><li> Figura. 1 mostra lo schema gating utilizzate per le analisi dei diversi sottogruppi di cellule T da un animale rappresentativo. I linfociti sono stati gated tramite forward scatter (FSC) rispetto side scatter (SSC), e poi vivere linfociti sono stati identificati sulla base di SSC e aqua-negativi popolazione. Le cellule T sono state poi identificato da CD3 espressione seguita dalla rilevazione dei CD4 + CD8-(cellule T CD4 +) e CD4-CD8 + (cellule T CD8 +) popolazioni nel CD3 + popolazione di cellule T. CD4 + e CD8 + T sono stati ulteriormente distinti in diversi sottoinsiemi sulla base di CD28 e CD95 espressione ingenua (Tn, CD28 + CD95-), la memoria centrale (Tcm, CD28 + CD95 +) e la memoria effettrici (Tem, CD28-CD95 +) cellule come descritto in letteratura 5, 10. </li><li> Figura. 2 mostra come i diversi sottoinsiemi di cellule CD4 + e CD8 + T è stata valutata la capacità funzionale in termini di produzione di citochine (INF-γ e / o IL-2) in risposta alla stimolazione con PMA e ionomicina (PMA + I). </li></ol> 5. Rappresentante FACS dati Composizione PBMC in macachi rhesus è stata determinata mediante analisi FACS. La figura 1 mostra lo schema gating utilizzate per le analisi dei diversi sottogruppi di cellule T da un animale rappresentativo. I linfociti sono stati gated tramite forward scatter (FSC) rispetto side scatter (SSC), e poi vivere linfociti sono stati identificati sulla base di SSC e aqua-negativi popolazione. Le cellule T sono state poi identificate da CD3 espressione. CD4 + CD8-(cellule T CD4 +) e CD4-CD8 + (cellule T CD8 +) popolazioni nel CD3 + popolazione di cellule T sono stati determinati. Come mostrato nella Figura 2, il CD4 + e CD8 + T sono stati ulteriormente distinti in diversi sottoinsiemi sulla base di CD28 e CD95 espressione ingenua (Tn, CD28 + CD95-), la memoria centrale (Tcm, CD28 + CD95 +) e la memoria effettrici (Tem, CD28-CD95 +) delle cellule. Figura 2A mostra INF-γ e IL-2 produzione in CD4 non stimolata e PMA / ionomicina stimolato + cellule T, e sottoinsiemi, e 2B figura mostra INF-γ e IL-2 produzione non stimolata e PMA / ionomicina stimolato cellule T CD8 +, e sottoinsiemi Notare i profili delle citochine distinte provocato dalla stimolazione in ogni sottogruppo. <img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Strategia di gating utilizzate per l&#39;analisi di citometria a flusso della composizione PBMC in rhesus macqaues. Si prega di <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="Figura 2a" border="0" /> <img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="Figura 2b" border="0" /> Figura 2. (A) profilo di produzione di citochine di cellule CD4 + T totale e sottoinsiemi. (B) profilo di produzione di citochine del totale delle cellule T CD8 + e sottoinsiemi. Si prega di <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">cliccare qui</a> per vedere una versione più grande di 2a, <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">o qui</a> per una versione più grande di figura 2b.

<ol>
	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

Soluzione e tamponi <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO) </li><li> RPMI-1640 medium (Hyclone laboratori, Logan, UT) </li><li> Siero fetale bovino (FBS), (Hyclone laboratori, Logan, UT) </li><li> L-glutamina (Sigma-Aldrich, St. Louis, MO) </li><li> Penicillina / streptomicina (Invitrogen, Carlsbad, CA) </li><li> Blu tripano reagente (Sigma-Aldrich, St. Louis, MO) </li><li> 50 mM Tris, pH 8,6, filtrate e conservare a 4 ° C </li><li> Dulbecco PBS (DPBS, Ca2/Mg2-free, Life Technology, Rockville, MD) </li><li> Flusso di tampone di lavaggio: supplemento DPBS con il 2% FBS, conservare a 4 ° C </li><li> BD Cytofix / Cytoperm Fixation / permeabilizzazione Solution Kit (BD Biosciences, San Jose, CA) </li></ul> Attivazione agenti <ul><li> Forbolo 12-miristato 13-acetato (PMA) (Sigma-Aldrich, St. Louis, MO) </li><li> Ionomicina (Sigma-Aldrich, St. Louis, MO) </li><li> Costimolazione mAb CD49d, clone 9F10 (BD Biosciences, San Jose, CA) </li><li> Purificate per affinità di anticorpi F (ab) 2 frammenti di capra anti-topo IgG (H + L) (Kierkegaard e Perry Laboratorio, Gaithersburg, MD) </li><li> Agenti di attivazione specifici potrebbero includere piscine peptide, peptide singolo, o proteine ​​intero </li></ul> Inibitori della secrezione di citochine <ul><li> Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO) </li><li> Plug Golgi può essere utilizzato in alternativa (BD Biosciences, San Jose, CA) </li></ul> Anticorpi <ul><li> Live / Dead risolvibile acqua kit fluorescenti macchia (Invitrogen, Carlsbad, CA) </li><li> CD3 PE-Cy7 (SP34-2), (BD Biosciences, San Jose, CA) </li><li> CD4 Pacific Blue (OKT4), (eBiosciences (San Diego, CA) </li><li> CD8 Alexa700 (RPA-T8), (BD Biosciences, San Jose, CA) </li><li> CD28 PerCP-Cy5.5 (L293), (BD Biosciences, San Jose, CA) </li><li> CD95 APC (DX2), (BD Biosciences, San Jose, CA) </li><li> IFN-FITC (B27), (BD Biosciences, San Jose, CA) </li><li> IL-2 PE (MQ1-17H12), (BD Biosciences, San Jose, CA) </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

Il modello di macaco rhesus è attualmente il miglior modello disponibile per l&#39;HIV-AIDS rispetto alla comprensione della patogenesi e per lo sviluppo di vaccini e terapie<sup&gt; 1,2,3</sup&gt;. Qui, descriviamo un metodo per l&#39;analisi dettagliate fenotipica e funzionale delle risposte immunitarie cellulari, in particolare la produzione di citochine intracellulari di CD4 + e CD8 + T, oltre che i sottoinsiemi memoria individuale. Abbiamo ottenuto precise misure quantitative e qualitative per la produzione di interferone gamma (INF-) e interleuchina (IL) -2 in entrambi i CD4 + e CD8 + T cellule del macaco rhesus PBMC stimolati con PMA più ionomicina (PMA + I). I profili delle citochine erano diverse nei diversi sottoinsiemi di celle di memoria. Inoltre, questo protocollo ci ha fornito la sensibilità per dimostrare anche le frazioni minori di specifico antigene CD4 + e CD8 + sottogruppi di cellule T nei campioni di PBMC da macachi rhesus immunizzati con un vaccino cocktail busta peptide HIV sviluppate nel nostro laboratorio. La tecnica di citometria a flusso multicolore è un potente strumento per identificare con precisione le diverse popolazioni di cellule T<sup&gt; 4,5</sup&gt; Con citochine capacità di produzione<sup&gt; 6</sup&gt; In seguito a stimolazione aspecifica o antigene-specifiche<sup&gt; 5,7</sup&gt;.

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Abbiamo dimostrato l&#39;utilità di citometria a flusso multicolore per informazioni dettagliate caratterizzazione fenotipica e funzionale del totale così come sottoinsiemi memoria di CD4 + e CD8 + T nei macachi rhesus, il modello ideale per l&#39;HIV / AIDS studi vaccino.

Multicolor Citometria a Flusso di analisi Cellular risposta immunitaria in macachi Rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

multicolor-flow-cytometry-analyses-cellular-immune-response-rhesus

Research

JoVE Journal

Immunology and Infection

21.0K Views.  MD Anderson Cancer Center - University of Texas. Abbiamo dimostrato l'utilità di citometria a flusso multicolore per informazioni dettagliate caratterizzazione fenotipica e funzionale del totale così come sottoinsiemi memoria di CD4 + e CD8 + T nei macachi rhesus, il modello ideale per l'HIV / AIDS studi vaccino.

Video: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

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 Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

L&#39;analisi dei Simian Immunodeficiency Virus specifici CD8<sup&gt; +</sup&gt; T-cellule in Rhesus macachi di peptide-MHC-I Tetramero colorazione

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

Analisi qualitativa e quantitativa della sinapsi Immune nel sistema umano utilizzando Imaging citometria a flusso

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

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 ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Cella singola quantificazione del mRNA e l'espressione di proteine di superficie CD4 infettati da Virus di immunodeficienza delle scimmie+ T cellule isolate da macachi Rhesus