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.

Das Protokoll hier vorgestellten wurde für die Verarbeitung von verschiedenen kultivierten Zelllinien (H1 und H9 hESCs, mESCs, MCF7, HEK 293, KBM5, HMEC, MCF10A) und primäre Gewebeproben (Maus-Knochenmark, embryonale Maus-Leber, Maus Erwachsenen erfolgreich eingesetzt Leber, die Maus-Tumor). Unabhängig von der Quelle, jedes Gewebe, das in einzelne Zellen dissoziiert werden kann, während der zelluläre Zustand bewahren sollte Zytometrie zur Analyse von Massen zugänglich sein, sondern kann eine Protokolloptimierung e...

Cytometry ermöglicht die gleichzeitige Messung mehrerer Antikörper Ziele in einer einzelnen Zelle Ebene über große Populationen von Zellen. In herkömmlichen fluoreszenzbasierten Durchflusszytometrie, die Anzahl der Parameter, die quantifiziert werden kann, wird durch spektrale Überlappung zwischen dem Emissionsspektrum von mehreren Fluorophoren begrenzt, die immer komplexer werdenden Kompensationsberechnungen wie die Anzahl der Parameter ansteigt erfordert. Diese Einschränkungen werden durch Massen adressiert Cytometry, wo Schwermetall-konjugierten Antikörper nachgewiesen werden, und durch Flugzeit (TOF) Massenspektrometrie quantifizieren stark die Anzahl der Parameter gesammelt gleichzeitig expandiert und ein hohes Dimensionsprotein Überfluß Profil für jede einzelne Zelle zu ergeben. Ein grundlegendes Verständnis der Funktionsweise des Massen Zytometrie Instrument ist für den Benutzer von Vorteil und kann für die Fehlersuche wesentlich sein. Für eine ausführliche Beschreibung der Abstimmung und eine Masse-Zytometrie Maschine ausgeführt wird,finden Sie im verwandten Manuskript 1. Kurz gesagt, wird eine Zellprobe mit einem Panel von Antikörpern , konjugiert Metallzielzelloberflächenmarker, cytoplasmatische Proteine, nukleäre Proteine, Chromatin-gebundene Proteine oder andere Epitope von Interesse (Figur 1i) markiert. Die markierten Zellen werden auf die Maschine geladen wird, entweder einen nach dem anderen durch manuelle Injektion oder mit Hilfe eines automatischen Probennehmer, dass die Proben von einer 96-Well-Platte. Die beladenen Zellen werden durch einen Zerstäuber injiziert wird , die einen Sprühnebel aus Flüssigkeitströpfchen erzeugt , um die Zellen einkapseln (Abbildung 1II). Dieses Spray wird so positioniert, dass die Zellen durch einen Argonplasmabrenner ionisiert werden. Diese Ionisierung erzeugt eine Partikelwolke aller konstituierenden Atome jeder Zelle (Abbildung 1III) umfasst. Da diese Partikelwolke zu dem Detektor bewegt werden geringe Atommassen Atome aus den Ionen mit hohen Masse durch einen Quadrupol-Massenfilter getrennt. Die verbleibenden Ionen mit hohen Masse weiter zu dem Detektor, wo der abundance jedes Isotop wird (Abbildung 1IV) quantifiziert. Die Rohdaten vom Detektor gesammelt werden, werden von den Massen Cytometry Gerätesoftware analysiert, um Zellereignisse zu identifizieren. Für jedes identifizierte Zelle Ereignis, das Signal in jedem Kanal detektiert wird quantifiziert und mit einem Ausgang .fcs Datei gespeichert. Die Massenkanäle verwendet für das Schwermetall konjugiert Nachweis von Antikörpern eine minimale spektrale Überlappung, die mit den meisten Beiträgen unter 1% von 0-4% liegt. Aufgrund dieses geringen Übersprechens zwischen den Kanälen, ist es in der Regel nicht erforderlich , die Daten mit einer Kompensationsmatrix vor der Analyse 2 zu transformieren. Allerdings sollte Sorgfalt bei der Gestaltung der experimenteller Gruppe von Antikörpern getroffen werden, um sicherzustellen, dass ein Antikörper mit einem Hochfluss-Epitop nicht zu einem Masse Kanal zugeordnet ist, das Signal an einen Kanal zu einem Low-Fülle-Antikörper, da dies zugewiesen trägt eine künstlich hohe Population in dem Kanal erzeugt den Signalbeitrag zu empfangen. <p class = „jove_content“&gt; Die genaue Vorbereitung von Proben für Massen Zytometrie-Analyse ist in hohem Maße abhängig von den Typen von Proben gesammelt und die experimentelle Hypothese getestet. Wir stellen Beispiele für zwei Protokolle für die Ernte verschiedene Arten von Zellen (menschliche embryonale Stammzellen) und primäre Zellen (Maus-Brusttumor-Epithelzelle Isolat). Wenn eine Masse Zytometrie Studie beginnen, ist es ratsam, einen kleinen Pilotversuch zum ersten führen, um sicherzustellen, dass alle Protokolle und Antikörpern genutzt werden arbeiten gut in der Hand des Prüfers. Dies ist besonders wichtig, wenn benutzerdefinierte sind konjugierte Antikörper verwendet werden, da die partielle Reduktionsreaktion während der Antikörper-Konjugation hat das Potenzial, Antikörperaffinität zu stören; Daher muss jeder individuelle Antikörper validiert werden empirisch Datengenauigkeit zu gewährleisten. Andere Überlegungen umfassen das Bestimmen, ob der Zelloberfläche Antikörper im Assay verwendet werden, werden ihre Epitope nach der Fixierung erkennen oder wenn sie need angewendet werden Zellen zu leben. Einige Fixierung verhindern richtige Färbung mit Antikörpern Zelloberfläche , wie bekannt ist , auftreten , die mit dem Peptid-MHC - Färbung 3, 4. Durchführen von Zelloberflächen - Antikörper - Färbung an lebenden Zellen kann für einige Antikörper erforderlich sein, aber dies schließt die Möglichkeit zur Antikörpermarkierung vor Strichkodierung da die meisten Strichcodes Verfahren nach der Fixierung durchgeführt werden , obwohl 5 Antikörper-basierte Strichkodierung 6, 7 ist eine Ausnahme. Bei der Bestimmung der Anzahl von Zellen für die Analyse vorbereitet werden, ist es wichtig zu wissen, dass nur ein Bruchteil der auf die Maschine geladen Zellen werden die Daten erfasst werden und produzieren. Dieser Verlust ist hauptsächlich auf Ineffizienzen, wenn der Zerstäuber um die Probe bei der Fackel Sprays. Der genaue prozentuale Verlust hängt von der Masse Zytometrie Maschinenaufbau und Zelltyp, sondern kann von 50 bis 85% liegen und solltebetrachtet, wenn das Experiment zu entwerfen. Dieses Protokoll wurde für 2x10 6 Zellen pro Probe optimiert, kann aber für die Verarbeitung von kleineren oder größeren Probengrößen angepasst werden. Dieses Protokoll kann von 1 × 10 6 bis 4 x 10 6, mit minimalen Modifikationen für Probengrößen verwendet werden , aber es ist wichtig , dass Fallzahl innerhalb eines Versuchs konsistent gehalten werden. Änderungen in der Zellzahl können die Intensität von Barcodes und Antikörper-Färbung beeinflussen und sollte in etwa konsistent gehalten werden, über Proben direkt miteinander verglichen werden. Einige Verfahren, wie tote Zellmarkierung und DNA-Markierung, sollen in der überwiegenden Mehrheit durchgeführt werden, wenn nicht alle experimentellen Designs. Die Kennzeichnung von toten Zellen in den Experimenten nur Oberflächenmarker Analyse kann mit Rh103 erreicht werden, aber Rh103 sollte für Experimente vermieden werden, in denen Permeabilisierung erforderlich ist, wie es in Färbung Verlust zur Folge hat. Für Experimente mit Permeabilisierung, ist es ratsam, Cisplatin zu nutzen Strichkodierung Proben können Antikörper Verbrauch verringern Erfassungszeit reduzieren und eliminieren Probe zu Probe Variabilität 9. Der Prozess der Strichkodierung beinhaltet einen einzigartigen probenspezifischen Code für alle Zellen Anwendung, die verwendet wird, um jede Zelle in seine Ursprungsprobe zuzuordnen. Nachdem die Proben Strichkodierung kann vor der Antikörperfärbung gepoolt werden, ein Verfahren, dass alle Proben gefärbt werden gleichermaßen gewährleistet. Um experimentelle Fehler zu minimieren, ist es ratsam, auf Barcode und bündelt alle Proben, die direkt miteinander verglichen werden. Derzeit gibt es mehrere Methoden für Strichkodierung Proben für Massen Zytometrie Analyse 5, 6, 7, von denen zwei hier vorgestellt werden (siehe unten). Mit derzeit erhältlichen reagents ist es möglich , die Fülle von 38 Antikörper Targets identifizieren Zellen in S-Phase 10, unterscheiden lebenden und toten Zellen und 8 messen zelluläre Niveaus von Hypoxie 11 in einem gemultiplexten Experiment mehrerer Proben zu quantifizieren. Diese Fähigkeit , hohe Parameter Einzelzellendaten für große Zellpopulationen zu sammeln , ermöglicht eine verbesserte Profilieren von sehr heterogenen Zellpopulationen und hat bereits eine Reihe von neuartigen biologischen Erkenntnissen 12, 13, 14, 15, 16 hergestellt. Destillieren der resultierenden komplexen Daten auf interpretierbare Informationen hat 18 die Verwendung von Computer - Algorithmen , einschließlich Spanning Tree Progression der Dichte Normalized Ereignisse (Pik) 17 und Visne erforderlich. Dieser Artikel stellt eine leicht zugänglicheÜberblick darüber, wie zu entwerfen und eine Masse-Zytometrie Experiment durchzuführen und stellt grundlegende Analyse von Massendaten-Zytometrie.

<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

Massen-Zytometrie verwendet mit Schwermetalletiketten konjugierte Antikörper, ein Ansatz, der stark erhöht die Anzahl der Parameter und Möglichkeiten für tiefe Analyse weit über hat, was möglich ist mit herkömmlichen fluoreszenzbasierten Durchflusszytometrie. Wie bei jeder neuen Technologie gibt es wichtige Schritte, die die zuverlässige Erzeugung von qualitativ hochwertigen Daten sicherzustellen. Dargestellt ist hier ein optimiertes Protokoll, das mehrere Techniken für die Verarbeitung von Zellproben für die Massen Zytometrie Analyse einbezieht. Die hier beschriebenen Methoden helfen dem Benutzer häufige Fehler zu vermeiden und konsistente Ergebnisse erzielen durch Variabilität zu minimieren, die zu ungenauen Daten führen kann. Um experimentelles Design zu informieren, die Gründe für optionale oder alternative Schritte in dem Protokoll und ihre Wirksamkeit bei der Aufdeckung neue Erkenntnisse in der Biologie des Systems wird untersucht, behandelt. Schließlich wird repräsentative Daten vorgelegt erwarteten Ergebnisse der Techniken presente zu illustrierenhier d.

Das Protokoll kann hier im Großen und Ganzen auf eine große Vielfalt von gezüchteten und primären Zellproben mit nur geringfügigen Modifikationen angewandt präsentiert werden. Je nach den Anforderungen des Experiments, kann es in einer modularen Art und Weise durchgeführt werden, wenn bestimmte Elemente, wie Barcodes oder Zelloberflächenfärbung, sind nicht erforderlich. Verwendetes in seiner Gesamtheit ermöglicht dieses Protokoll, um die Herstellung von Massen gemultiplexten Cy...

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.

Probenvorbereitung für die Massen Cytometry-Analyse

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

Serie: 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-Probenvorbereitung: Die Ultra Thin-Layer-Methode

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

Forschung

Biologie

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 Blot: Probenvorbereitung zur Detektion

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.

Probenvorbereitung für Single Virion Atomic Force Microscopy und 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 ...

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.

Techniken für die Analyse der extrazellulären Vesikeln mittels Durchflusszytometrie

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.

Ein alternativer Ansatz für die Probenvorbereitung mit geringer Zellzahl für die TEM-Analyse

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.

Probenvorbereitung und Bildgebung von Exosomen von Transmissions-Elektronenmikroskopie

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.

Miniaturisierte Probenvorbereitung für Transmissions-Elektronenmikroskopie

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.

Spaltöffnungen Tape-Peel: Ein verbessertes Verfahren zur Wache Zelle Probenvorbereitung

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.

Probenvorbereitung für Masse-Massenspektrometrie-basierte Proteomics Analyse der okulären Mikrogefäßen

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.

Probenvorbereitung für metabolisches Profiling mittels MALDI-Massenspektrometrie-Bildgebung

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