Work in the Barton lab was supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT RP110471). This research was supported in part, by a training grant fellowship for Ryan L. McCarthy from the National Institutes of Health Training Program in Molecular Genetics 5 T32 CA009299. The core facility that maintains and runs the mass cytometry machine is supported by CPRIT grant (RP121010) and NIH core grant (CA016672).

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The authors have nothing to disclose.

Il protocollo presentato qui è stato impiegato con successo per il trattamento di varie linee in coltura di cellule (H1 e H9 hESC, mESCs, MCF7, HEK 293, KBM5, HMEC, MCF10A) e campioni di tessuti primari (midollo osseo di topo, il mouse del fegato embrionale, il mouse per adulti fegato, tumore mouse). Indipendentemente dalla fonte, qualsiasi tessuto che può essere dissociato in singole cellule preservando lo stato cellulare dovrebbe essere suscettibile di analisi mediante citometria di massa, ma può richiedere alcuni ...

Citometria consente la misurazione simultanea di più target anticorpali a livello di singola cellula attraverso grandi popolazioni di cellule. Nel tradizionale citometria di flusso basato sulla fluorescenza, il numero di parametri che possono essere quantificate è limitato dalla sovrapposizione spettrale tra lo spettro di emissione dei fluorofori multipli, che richiede calcoli di compensazione sempre più complessi come il numero di parametri aumenta. Queste limitazioni sono rivolte in massa citometria, dove vengono rilevati e quantificati mediante tempo di volo (TOF) spettrometria di massa per espandere notevolmente il numero di parametri raccolti simultaneamente e produrre un alto dimensionale profilo proteico-abbondanza per ogni singola cella anticorpi metallo coniugati pesanti. Una conoscenza di base del funzionamento della massa citometria strumento è vantaggioso per l&#39;utente e può essere essenziale per la risoluzione dei problemi. Per una descrizione più approfondita di messa a punto e l&#39;esecuzione di una macchina citometria di massa,consultare il relativo manoscritto 1. Brevemente, un campione di cellule è etichettato con un pannello di metallo anticorpi coniugati destinati marcatori di superficie cellulare, le proteine citoplasmatiche, proteine nucleari, proteine cromatina-bound o altri epitopi di interesse (Figura 1i). Le cellule marcate vengono caricate sulla macchina, uno alla volta mediante iniezione manuale o mediante un autocampionatore che i campioni da una piastra a 96 pozzetti. Le cellule caricate vengono iniettate attraverso un nebulizzatore, che genera uno spruzzo di goccioline di liquido che incapsulano le cellule (Figura 1ii). Questo spray è posizionato in modo che le cellule vengono ionizzati da una torcia al plasma di argon. Questo ionizzazione genera una nube di particelle comprendente tutti gli atomi costituenti ciascuna cella (Figura 1iii). Poiché questa nube particella viaggia verso il rivelatore, bassi atomi di massa atomica sono separati dalle alte ioni massa da un filtro di massa a quadrupolo. I restanti alti ioni di massa continuano al rivelatore, dove l&#39;abundance di ciascun isotopo viene quantificato (Figura 1iv). I dati grezzi raccolti dal rivelatore, vengono analizzati dal software dello strumento citometria di massa per identificare gli eventi cellulari. Per ciascun evento cella identificata, il segnale rilevato in ciascun canale viene quantificata e salvata in un file un&#39;uscita .fcs. I canali di massa utilizzati per rilevare gli anticorpi metallo pesante coniugati presentano minima sovrapposizione spettrale, che va da 0,4% con la maggior parte dei contributi inferiori all&#39;1%. A causa di questa cross-talk tra i canali, è generalmente necessario trasformare i dati con una matrice di compensazione prima dell&#39;analisi 2. Tuttavia, occorre prestare attenzione durante la progettazione del pannello sperimentale di anticorpi per garantire che un anticorpo con un epitopo alta abbondanza non è assegnato a un canale di massa che contribuisce segnale ad un canale assegnato ad un anticorpo a bassa abbondanza, in quanto ciò può creare artificialmente elevati della popolazione nel canale di ricezione del segnale del contributo. <p class = &quot;jove_content&quot;&gt; La preparazione esatta di campioni per analisi di citometria di massa dipende fortemente i tipi di campioni che vengono raccolti e l&#39;ipotesi sperimentale in fase di test. Forniamo esempi di due protocolli per la raccolta diversi tipi di cellule (cellule staminali embrionali umane) e cellule primarie (topo tumore mammario isolare cellule epiteliali). Quando si inizia uno studio citometria di massa, si consiglia di effettuare prima un piccolo esperimento pilota per garantire che tutti i protocolli e gli anticorpi per essere utilizzati stanno lavorando bene nelle mani del ricercatore. Questo è particolarmente importante quando devono essere utilizzati anticorpi coniugati personalizzati, come la reazione di riduzione parziale durante la coniugazione anticorpo ha il potenziale di distruggere affinità anticorpale; Pertanto, ogni anticorpo personalizzato deve essere convalidata empiricamente per assicurare l&#39;accuratezza dei dati. Altre considerazioni includono la determinazione se gli anticorpi superficie cellulare da utilizzare nel saggio riconoscerà loro epitopi dopo fissazione o se NEEd da applicare per vivere le cellule. Alcuni fissaggio può impedire il corretto colorazione con anticorpi superficie cellulare come noto verifica con peptide-MHC colorazione 3, 4. Esecuzione di superficie cellulare colorazione anticorpale su cellule vive può essere necessario per alcuni anticorpi, ma esclude la possibilità di codici a barre prima dell&#39;etichettatura anticorpo come maggior parte dei metodi di codici a barre vengono eseguiti dopo fissazione 5 sebbene codici a barre a base di anticorpi 6, 7 è un&#39;eccezione. Nel determinare il numero di cellule da preparare per l&#39;analisi, è importante sapere che solo una frazione delle cellule caricate sulla macchina sarà rilevato e produrre dati. Questa perdita è dovuta in primo luogo a inefficienze quando il nebulizzatore spray campione al cannello. La perdita percentuale esatta dipende dalla massa citometria configurazione della macchina e tipo cellulare, ma può variare da 50-85% e deve essereconsiderati quando si progetta l&#39;esperimento. Questo protocollo è stato ottimizzato per 2x10 6 cellule per campione, ma può essere adatta a lavorare campioni di dimensioni più piccole o più grandi. Questo protocollo può essere utilizzato con minime modifiche per dimensioni del campione da 1 x 10 6 a 4 x 10 6, tuttavia è fondamentale che la dimensione del campione essere mantenuta costante in un esperimento. Cambiamenti nel numero di cellule possono influenzare l&#39;intensità dei codici a barre e colorazione degli anticorpi ed essere comunque ridotti all&#39;incirca costante attraverso campioni da confrontare direttamente. Alcune procedure, come l&#39;etichettatura di cellule morte e l&#39;etichettatura del DNA, devono essere effettuate nella stragrande maggioranza, se non tutti i disegni sperimentali. L&#39;etichettatura di cellule morte in esperimenti analizzare solo marcatori di superficie può essere ottenuta utilizzando Rh103, ma Rh103 dovrebbe essere evitato per esperimenti in cui è richiesta permeabilizzazione come si verifica una perdita di colorazione. Per gli esperimenti che coinvolgono permeabilizzazione, si consiglia di utilizzare cisplatino Campioni codici a barre possono ridurre il consumo di anticorpi, ridurre il tempo di acquisizione ed eliminare campione a campione variabilità 9. Il processo di barcoding comporta l&#39;applicazione di un codice specifico di esempio unico per tutte le celle, che viene utilizzato per assegnare ogni cella per il campione di origine. Dopo barcoding i campioni possono essere riuniti prima della colorazione anticorpale, un processo che assicura tutti i campioni sono macchiati ugualmente. Per ridurre al minimo errore sperimentale, è prudente codice a barre e piscina i campioni che devono essere direttamente confrontate tra di loro. Attualmente esistono diversi metodi per codici a barre dei campioni per l&#39;analisi di citometria di massa 5, 6, 7, due dei quali sono qui presentati (vedi sotto). Con Reagen attualmente disponibilits è possibile quantificare l&#39;abbondanza di 38 bersagli degli anticorpi, identificare le cellule in fase S 10, distinguere le cellule vive e morte 8 e misurare i livelli cellulari di ipossia 11 in un esperimento multiplex di campioni multipli. Questa capacità di raccogliere dati monocellulari alta parametro per grandi popolazioni cellulari consente una migliore profilatura di popolazioni cellulari altamente eterogenee e ha già prodotto una serie di dati biologici nuovi 12, 13, 14, 15, 16. Distillando i dati complesso risultante per informazioni interpretabili ha richiesto l&#39;uso di algoritmi di calcolo includendo Spanning Tree progressione della densità Eventi normalizzati (SPADE) 17 e 18 viSNE. Questo articolo presenta una accessibilipanoramica di come progettare e realizzare una massa citometria esperimento e introduce analisi di base della massa citometria dati.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>Stem Cell technologies</td>
			<td>05850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>DMEM F-12</td>
			<td>ThermoFisher</td>
			<td>11330-032</td>
			<td>Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell technologies</td>
			<td>07920</td>
			<td>Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Equitech</td>
			<td>BAH62</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell ID Pt194</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt195</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt196</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-400-040-M250</td>
			<td>Soluble to 25mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Cell-ID 20-plex Pd Barcoding Kit</td>
			<td>Fluidigm</td>
			<td>201060</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Iodo-2&#39;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>I7125</td>
			<td>Soluble to 74mg/ml in 0.2N NaOH</td>
		</tr>
		<tr>
			<td>Rhodium 103 intercelating agent</td>
			<td>Fluidigm</td>
			<td>201103A</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>BP1105-4</td>
			<td>Chill at -20&#176;C before use</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml round bottom tubes</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml 35um filter cap tubes</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>EQ four element calibration beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase Type I-S</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel #10</td>
			<td>Sigma-Aldrich</td>
			<td>Z69239</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation for mass cytometry analysis

Massa utilizza citometria anticorpi coniugati con le etichette di metalli pesanti, un approccio che ha notevolmente aumentato il numero di parametri e opportunità per l&#39;analisi pozzo profondo di quanto sia possibile con i tradizionali flusso basato sulla fluorescenza citometria. Come per ogni nuova tecnologia, ci sono passaggi critici che aiutano a garantire la generazione affidabile di dati di alta qualità. Qui presentata è un protocollo ottimizzato che comprende molteplici tecniche per il trattamento di campioni di cellule per l&#39;analisi di citometria di massa. I metodi descritti qui aiuterà l&#39;utente a evitare problemi comuni e raggiungere risultati coerenti minimizzando variabilità, che può portare a dati accurati. Per informare disegno sperimentale, la logica alla base operazioni opzionali o alternative nel protocollo e la loro efficacia nello scoprire nuove scoperte nella biologia del sistema oggetto di indagine è coperto. Infine, i dati rappresentativi è presentato per illustrare i risultati attesi dalle tecniche Presented qui.

Il protocollo presentato qui può essere ampiamente applicata ad una vasta gamma di campioni di cellule in coltura e primario con modifiche solo minori. A seconda delle esigenze dell&#39;esperimento, può essere eseguita in modo modulare quando alcuni elementi, come codici a barre o superficie cellulare colorazione, non sono necessarie. Utilizzato nella sua interezza questo protocollo consente la preparazione di massa multiplato citometria campioni etichettati per identificazione popolaz...

Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Sample Preparation for Mass Cytometry Analysis

This article describes the collection and processing of samples for mass cytometry analysis.

Preparazione del campione per l&#39;analisi di massa Citometria

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

sample-preparation-for-mass-cytometry-analysis

Research

JoVE Journal

Biology

16.0K Views.  The University of Texas MD Anderson Cancer Center. This article describes the collection and processing of samples for mass cytometry analysis.

Video: Sample Preparation for Mass Cytometry Analysis

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Preparazione del campione: il metodo ultra sottile strato

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Ricerca

Biologia

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: la preparazione dei campioni di rilevamento

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Preparazione del campione per microscopia a forza atomica Virion singola e imaging a fluorescenza a super risoluzione

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Tecniche per l&#39;Analisi dei extracellulare vescicole citometria a flusso

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Un approccio alternativo per la preparazione del campione con un basso numero di cellule per l&#39;analisi TEM

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparazione del campione e la formazione immagine di esosomi da microscopia elettronica di trasmissione

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Preparazione del campione miniaturizzati per microscopia elettronica di trasmissione

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Gli stomi nastro-Peel: Un metodo migliore per la preparazione del campione di cellule di guardia

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Preparazione del campione per l'analisi di proteomica massa-spettrometria-basato di microvasi oculari

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Preparazione del campione per la profilazione metabolica utilizzando la spettrometria di massa MALDI

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...