Questo lavoro è stato sostenuto dal Premio Innovazione Eagles (Fraternal Order of Eagles) e dalla Fondazione Mayo.

** Questo protocollo video si basa su una pubblicazione associata 1: ad alta sensibilità di rilevazione e analisi quantitativa dei nativi interazioni proteina-proteina e complessi multiproteici mediante citometria a flusso. Adam G. Schrum, Diana Gil, Elaine P. Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang WA Schamel, Ed. Palmer. Scienza STKE 2007 (389): PL2, 5 giugno 2007, [DOI: 10.1126/stke.3892007pl2]. <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">Clicca qui per vedere questa pubblicazione</a> . Prima di partire, Preparare le seguenti soluzioni acquose archivi: <table><tbody><tr><td> MES Accoppiamento buffer: </td><td> Conservare a 25 ° C </td></tr><tr><td> MES, pH 6,0 </td><td> 50 mM </td></tr><tr><td> EDTA </td><td> 1 mM </td></tr><tr><td> Tempra / Blocco / Storage (QBS) Buffer: </td><td> Conservare a 4 ° C </td></tr><tr><td> BSA </td><td> 1% </td></tr><tr><td> Di sodio azide </td><td> 0,02% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr><tr><td> FCM buffer: </td><td> Conservare a 4 ° C </td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 100 mM </td></tr><tr><td> Di sodio azide </td><td> 0,02% </td></tr><tr><td> FBS </td><td> 5% </td></tr><tr><td> PBS, pH 7,4 </td><td> 1x </td></tr></tbody></table> Preparare immediatamente prima dell&#39;uso: <table><tbody><tr><td> EDAC-MES: </td><td> Sciogliere 50 mg / ml polvere EDAC nel tampone MES accoppiamento </td></tr><tr><td> Digitonina soluzione (2% w / v): </td><td> Sciogliere in polvere digitonina dH 2 O da riscaldamento a 95 ° C per 5 minuti poi il raffreddamento su ghiaccio </td></tr><tr><td> Lysis buffer: </td><td></td></tr><tr><td> Tris, pH 7,4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 150 Mm </td></tr><tr><td> Digitonina soluzione </td><td> 1% </td></tr><tr><td> Inibitori della proteasi </td><td> 1x </td></tr><tr><td> Tenere su ghiaccio </td><td></td></tr></tbody></table> 1. Accoppiamento di mAb di Perle <ol><li> Determinare la concentrazione di perline dallo stock acquistato da diluire in PBS e contando con un emocitometro. Inizia con una diluizione 1:10000 di perline per il conteggio. </li><li> Pipettare 18 x 10 6 perle in un 1,5 mL provetta. </li><li> Lavare le perline 2 o 3 volte in 0,5-1,0 mL di tampone di accoppiamento MES, centrifugazione a 20.000 g per 3 minuti a 25 ° C dopo ogni lavaggio. </li><li> Risospendere le sfere in 50 microlitri Buffer accoppiamento MES. </li><li> Attivare i gruppi carbossilici sulla perle aggiungendo 20 l di preparati al momento EDAC-MES. </li><li> Mescolare delicatamente per 15 minuti a 25 ° C manualmente pipettando su e giù. </li><li> Lavare le perline attivate 2 o 3 volte in 0,5 a 1,0 ml di PBS, centrifugazione a 20.000 g per 3 minuti a 25 ° C dopo ogni lavaggio. </li><li> Risospendere le sfere attivato in 50 microlitri di PBS. </li><li> Aggiungere 50 microlitri della IP mAb (a 0,2-1,0 mg / ml concentrazione stock) alla soluzione attivata tallone. </li><li> Mescolare per 3 o 4 ore a 25 ° C posizionando il tubo su una vibrante shaker. Agitare sufficiente per evitare sedimentazione delle perline sul fondo della provetta. </li><li> Lavare mAb-perline accoppiate 2 o 3 volte in 0,5 a 1,0 ml di PBS, centrifugazione a 20.000 g per 3 minuti a 25 ° C dopo ogni lavaggio. </li><li> Risospendere le sfere in 100 microlitri Buffer QBS. Questi possono essere conservato a 4 ° C. </li><li> Sospendere le perline bene, diluire 1:200-1:10,000 in PBS e contare con un emocitometro. La concentrazione deve essere misurato per ciascun lotto preparativa, in modo che il numero di perline usato in ogni esperimento IP o pull-down può essere controllato con precisione. </li></ol> 2. Post-nucleare Preparazione del lisato e Ip Il metodo di lisi e le condizioni di lisi ottimale dipenderà dal tipo di cellula e le interazioni proteina-proteina in esame. Un certo numero di protocolli esistenti e può essere modificato per l&#39;applicazione. <ol><li> Lyse 20 x 10 6 &#39;piccolo&#39; cellule, come i linfociti, o 5 x 10 6 &#39;grandi&#39; le cellule, come i macrofagi o cellule tumorali, in 100 microlitri di buffer di lisi fresco in un 1,5 mL provetta per 20 minuti sul ghiaccio. Scala il volume lisi, se necessario. </li><li> Per rimuovere i nuclei e insolubili detriti cellulari, centrifugare il lisato a 20.000 g per 10 minuti a 4 ° C. Tenere il supernatante e scartare il pellet. </li><li> Aggiungere 0,5 x 10 5 a 2,5 x 10 5 mAb-perline accoppiate al lisato chiarificato, pipettare delicatamente per mescolare. Il volume minimo che usiamo pereseguire un singolo IP è di 5 microlitri. </li><li> Mettere su una ruota in movimento verticale 4 ore per notte in una stanza fredda. Impostare a velocità sufficiente per evitare che le perle di assestamento. È accettabile che il liquido di basso volume IP di rimanere nel fondo della provetta durante la rotazione. </li></ol> 3. Probing di Bead-capture di proteine ​​con Fluorocromo anticorpi coniugati <ol><li> Lavare le perline IP due volte in 0,2 a 1,0 ml ghiacciata buffer FCM, centrifugazione a 20.000 g a 4 ° C per 3 minuti dopo ogni lavaggio. </li><li> Risospendere le sfere in 100 ul FCM tampone e aliquota 20 l in ciascuno dei cinque o più di 1,5 mL microprovette o pozzi di un camino con fondo piatto. </li><li> Aggiungi coniugato a un fluorocromo anticorpi monoclonali ai campioni. Eseguire la FCM macchia secondo le istruzioni del fornitore o in base a concentrazioni empiricamente determinato. Come punto di partenza, aggiungere 0,2 a 1 microlitri di soluzione madre di anticorpi (a 0,2-1,0 mg / ml) al tubo o bene e incubare per 40 minuti sul ghiaccio. </li><li> Lavare sondato perline in tubi da 1,5 ml due volte in 1,0 ml ghiacciata buffer FCM, centrifugazione a 20.000 g a 4 ° C per 3 minuti dopo ogni lavaggio. Rimuovere delicatamente il supernatante dopo ogni lavaggio, facendo attenzione a non disturbare le perline. Per un camino a fondo piatto, lavare due volte con 0,2 mL ghiacciata buffer FCM, centrifugazione a 1000g per 5 minuti a 4 ° C dopo ogni lavaggio. Una pipetta multicanale è utile per questo passo. Per rimuovere il surnatante da perle in un camino-piastra inferiore, &#39;sfogliare&#39; il piatto una volta, poi, mentre ancora a testa in giù, ogni macchia gocciola dai bordi dei pozzi. Il volume residuo in ciascun pozzetto, contenente le perline, sarà di circa 20 l. </li><li> Risospendere le sfere in 200 Buffer FCM microlitri per campione. Trasferimento a tubi etichettati FACS. I campioni sono ora pronti per FCM. </li></ol> 4. FCM Acquisizione Le perle CML descritte in questo protocollo sono da 3 a 5 micron di diametro, circa la metà del diametro di un linfocita del mouse quiescente. Pertanto, può essere necessario aumentare manualmente il Forward Scatter (FSC) guadagno amplificatore e il Side Scatter (SSC) di tensione in modo che la popolazione di eventi tallone di registrare su scala. Individuale perline IP dovrebbero formare un unico popolo strettamente cluster. Le impostazioni e cancello deve essere regolato per escludere doppietti tallone e detriti. <ol><li> Prima di perline in esecuzione standard o tallone, rimuovere il manicotto di contenimento goccia che circonda l&#39;ingresso del campionatore su alcuni citometri. Che il liquido guaina a gocciolare tra i campioni, in modo da cancellare l&#39;ingresso ed impedire perline di essere riportati da un campione all&#39;altro. </li><li> Usa perle senza etichetta, un controllo negativo di fluorescenza, e particelle di calibrazione arcobaleno (RCPs), un controllo positivo di fluorescenza, per regolare le impostazioni in modo che entrambi gli estremi negativi e positivi fluorescenza appare contemporaneamente sul log-scala asse delle ascisse. Questo assicura che la fluorescenza da campioni sperimentali saranno inoltre on-scala. Si noti che RCPs sono più piccole rispetto alle perle CML, in modo da FSC e parametri SSC, nonché la posizione cancello, potrebbe essere necessario essere temporaneamente modificato per quel campione. </li><li> Torna a senza etichetta-tallone impostazioni una volta di calibrazione con RCPs è completa. Se solo una sonda fluorescente per campione è usato, senza compensazione della fluorescenza è necessario. In generale, si sonda complessi multiproteici con un solo fluorocromo-coniugati mAb per campione FCM, e prendiamo in esame l&#39;interazione partner più dalla colorazione parallelo campioni di cordone con anticorpi monoclonali specifici per le subunità diverse. </li><li> Acquisire e salvare i file di dati di fluorescenza. L&#39;analisi può essere effettuata utilizzando il software citometria a flusso come CellQuest, FlowJo, o CFlow. </li></ol> 5. Rappresentante Risultati <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="Figura 1" /> Figura 1. IP-FCM per TCR/CD3 complesso multiproteico. Cellule T dal ceppo di topi BALB / c sono state lisate in 1% digitonina, e il lisato è stato sottoposto a IP-FCM con anti-CD3ε perline. I complessi catturato conteneva quantità significative di TCR-β (regione viola) e co-associati CD3-ε (verde), ma vicino ai livelli di fondo (definito dalla sonda immunoglobuline irrilevante, rosa traccia) di altre proteine, come Thy1.2, CD45, o H-2K (d) (marrone, arancio, blu e tracce, rispettivamente). Vedi simili risultati precedentemente pubblicati 1-6.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

Informazioni sulle interazioni proteina-proteina è molto importante per l&#39;analisi di molti processi cellulari come la trasduzione del segnale, la maturazione lignaggio, progressione del ciclo cellulare, apoptosi e cascate. IP-FCM fornisce un modo rapido, quantitativa e sensibile di esaminare l&#39;interazione delle proteine ​​e definiscono i membri di complessi multiproteici nella loro conformazione nativa. Sfere possono essere accoppiati e incubato con lisati cellulari in un giorno e possono essere esaminati e analizzati il ​​giorno successivo. A 96 pozzetti formato piastra permette un gran numero di campioni da analizzare in una sola volta per fornire efficienti di raccolta dati a fini statistici o di screening. Utilizzando perline fluorescenti standard, il numero di proteine ​​catturate da ogni perla può essere stimata. Molto materiale di origine poco è necessario per la cattura e il rilevamento, in modo da campioni limitati e scarsa analiti possono ancora essere analizzati per le interazioni multiple. Sebbene IP-FCM non richiede manipolazione genetica, epitopo-tagging, denaturazione, in vitro o miscelazione di proteine ​​in modo non fisiologico ambiente, può essere accoppiato con queste e altre tecniche, che lo rende uno strumento valido e accessibile con applicabilità a molti sistemi biologici. Risoluzione dei problemi: Molti IP-FCM esperimenti di generare dati utili interazioni proteiche la prima volta che sono tentato. Tuttavia, l&#39;ottimizzazione di IP-FCM può migliorare la coniugazione di anticorpi a perline, catturare le proteine ​​complesse, e la sonda fluorescente vincolanti. L&#39;efficienza di coniugazione degli anticorpi IP può essere determinato dal sondaggio perline accoppiate direttamente con un anticorpo anti-immunoglobulina. Se questo rendimento è basso, aumentando la concentrazione di anticorpi durante la reazione di accoppiamento può permettere anticorpi più IP ad allegare ad ogni tallone. Questo può aumentare la capacità di legame del lotto IP tallone, con conseguente acquisizione maggiore e la rilevazione di analiti. Altre molecole contenenti ammine primarie (ad esempio tris, albumina di siero bovino) non deve essere presente durante la reazione di accoppiamento, in quanto questi possono competere con i mAb per il fissaggio tallone e risultato a basso accoppiamento mAb. Se coniugazione di mAb di perline si rivela problematica per altre ragioni, perline può invece essere accoppiato ad avidina / streptavidina, e gli anticorpi biotinilati può successivamente essere non un legame covalente e utilizzati per immunprecipitation. Nonostante la coniugazione degli anticorpi buona, la rilevazione iniziale di perline associate fluorescenza può essere bassa. In primo luogo, la cattura complesso si può essere bassa. Poiché IP-FCM dipende dalla concentrazione di analiti, aumentando il numero di cellule lisate per unità di volume lisi può aumentare la cattura degli analiti e la rilevazione. Inoltre, l&#39;acquisizione può essere migliorata riducendo il numero di perline IP incubati con il lisato, che distribuisce i complessi catturati attraverso un minor numero di perle, e si traduce in un aumento medio per fluorescenza tallone quando sondato. In secondo luogo, è possibile che l&#39;accesso degli anticorpi sonda ai complessi catturato è stericamente ostacolato dalla anticorpi immunoprecipitating. In questo caso, il problema può essere affrontato utilizzando anticorpi diversi per catturare e / o sonda.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
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ip-fcm:  immunoprecipitation detected by flow cytometry

Immunoprecipitazione rilevata mediante citometria di flusso (IP-FCM) è un metodo efficace per il rilevamento e la quantificazione interazioni proteina-proteina. Il principio di base che si estende su panino ELISA, in cui l&#39;analita catturato primario può essere rilevato insieme con altre molecole associate fisicamente all&#39;interno di complessi multiproteici. La procedura prevede accoppiamento covalente di microsfere in lattice di polistirene con immunoprecipitating anticorpi monoclonali (mAb) specifico per una proteina di interesse, incubando queste perle con lisati di cellule, sondando complessi di proteine ​​catturate con fluorocromo-coniugati sonde, e analizzando perline associati fluorescenza mediante citometria di flusso. IP-FCM è estremamente sensibile, permette l&#39;analisi delle proteine ​​nella loro nativa (non denaturato) dello stato, ed è suscettibile di entrambe le analisi semi-quantitativa o quantitativa. Come ulteriori vantaggi, IP-FCM non richiede manipolazione genetica o attrezzature speciali, che non sia un citofluorimetro, e può essere facilmente adattato per applicazioni ad elevato throughput.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

L&#39;IP-FCM metodo viene presentato, che permette una sensibile, robusto, la valutazione biochimica di natale interazioni proteina-proteina, senza la necessità di ingegneria genetica o di campioni di grandi dimensioni.

IP-FCM: Immunoprecipitazione rilevato mediante citometria di flusso

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.3K Views.  Mayo Clinic. L'IP-FCM metodo viene presentato, che permette una sensibile, robusto, la valutazione biochimica di natale interazioni proteina-proteina, senza la necessità di ingegneria genetica o di campioni di grandi dimensioni.

Video: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Ricerca

Biologia

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Misurazione quantitativa della traslocazione GLUT4 sulla membrana plasmatica mediante citometria di flusso

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Immunolocalizzazione della membrana esterna proteine ​​mediante citometria di flusso di mitocondri isolati

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Thermo flusso LED - La combinazione Optogenetics con citometria a flusso

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Misurazione di alto-rendimento di Dictyostelium discoideum Macropinocitosi tramite flusso Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...