Teil 1: Vorbereitung der mikrofluidischen Kanälen eines BIOFLUX Platte. <ol><li> Vor dem Ausführen der Durchflusszelle Experiment, muss man die mikrofluidischen Kanälen mit einem Protein-Beschichtung von Interesse vorzubereiten. In diesem Fall, Kollagen I verwendet werden. Jeder Kanal verfügt über einen Einlass und einen Auslass gut. Für diese Platte, die Einlass und die linke und Fütterung des Kanals und der Ausgang auch auf der rechten Seite. </li><li> Verdünnen Sie die Kollagen I (5mg/ml Lager) Beschichtung auf 200μg/ml Konzentration in 0,02 M Essigsäure. Man wird 20 &amp;mgr; l für jeden Kanal verwendet müssen. Mix von sanften triteration mit einer Mikropipette Spitze. </li><li> Add 20 ul Beschichtung auf dem jeweiligen Kanal verwendet werden. Man muss die Flüssigkeit in dem inneren Stempel der Steckdose und Fütterung der mikrofluidischen Kanal von Interesse mit einer Mikropipette zu verzichten. Vermeiden Sie die Einführung Blasen, nicht drücken die Luft aus der Pipette. Fügen Sie einen Kanal ohne Kollagen für eine nicht Kollagen-Steuerung (negative Kontrolle für die Adhäsion und Aggregation). </li><li> Bringen Sie die Schnittstelle an die Platte, indem Sie zuerst den Finger Anziehen der 4 Schrauben und dann, sobald sie alle gesetzt sind, verwenden Sie das Drehmoment Fahrer komplett anziehen. Das Drehmoment-Treiber klicken, wenn es die maximale Dichtigkeit erreicht. </li><li> Mit manuellen Modus in den BIOFLUX Software gelten Perfusion, um die Kanäle von Interesse an 2dyn/cm 2-aus der Steckdose gut. Hier muss man für das Gegenteil inneren Punsch mit Flüssigkeit gefüllt werden sehen. Dies wird einige Minuten dauern. Man kann entweder passen Sie dieses auftreten mit einem Low-Power-Ziel auf einem Mikroskop (4X) und die Suche nach dem Einlauf gut oder indem Sie die Platte und sucht in den Einlass Brunnen von unten. Man sollte zuerst eine winzige Kügelchen von Flüssigkeit, die langsam füllt den inneren Punsch. Sobald der Einlass inneren Punsch gefüllt ist, zu stoppen Perfusion auf einmal, indem Sie Halt in der Software. </li><li> Die Platte bei Raumtemperatur für eine Stunde. </li><li> Entfernen Sie die Schnittstelle und 1 ml PBS (plus Ca 2 + / Mg 2 +) zum Auslass gut. Starten Perfusion aus der Steckdose und am 2dyn/cm 2. Weiter Perfusion für 10 Minuten. Stop-Perfusion und entfernen Sie die Schnittstelle. </li><li> Entfernen Sie überschüssiges PBS aus beiden Ein-und Auslass Brunnen - nie flüssiges, dass der innere Stempel füllt. Blockieren Sie die Kanäle mit Blocking-Lösung (0,5% v / v BSA in PBS (plus Ca 2 + / Mg 2 +)). 1 ml Blocking-Lösung in jede Steckdose gut genutzt werden und perfuse aus der Steckdose und am 2dyn/cm 2. Weiter Perfusion für 10 Minuten. Stop-Perfusion und entfernen Sie die Schnittstelle. Kanäle bis zu diesem Schritt bereit sind den ganzen Tag genutzt werden. </li><li> Vor der Zugabe Blut, entfernen Sie die Flüssigkeit auf beiden Seiten des Kanals mit Ausnahme der inneren Stempel. </li></ol> Part 2. Vorbereiten des Blutes mit GPIIb / IIIa hemmende Antikörper. <ol><li> Während das Kollagen in den Kanälen Inkubation ist, muss man vorbereiten Vollblut. Man sollte folgen der Biosicherheit Vorschriften diktiert von seinem / ihrem Institut im Umgang mit menschlichem Blut. </li><li> Frisches menschliches Blut (von Nüchtern) in Natrium-Citrat als Antikoagulanz gezogen sollten innerhalb von 3 Stunden nach der Entnahme verwendet werden. </li><li> Bereiten Sie das Blut durch Zugabe von 4 &amp;mgr; m Calcein AM. Bereiten Sie ein 4mm Lager von Calcein AM in DMSO und fügen Sie Blut in einer Verdünnung von 1 / 1000 v / v. Mix durch vorsichtiges Schwenken. </li><li> Dispense 1 ml der Calcein AM gekennzeichnet Blut in 10 - 1,5 ml Röhrchen. Add GPIIb / IIIa-Inhibitor-Antikörper gegen jeden in der gewünschten Verdünnung (das Molekulargewicht von IgG ~ 150kDa). Ein Beispiel Verdünnungsreihe von 1200nm bis 9Nm und beinhaltet eine nicht-Antikörper-Steuerung und einem nicht verwandten Antikörper-Kontrolle. Eine ausgezeichnete positive Kontrolle für die Inhibition würde ReoPro werden (Abciximab, Eli Lilly) in einer Konzentration von 20ug/ml. Mix durch vorsichtiges Schwenken. </li><li> Inkubieren bei Raumtemperatur für 1 Stunde. Mix durch vorsichtiges Schwenken alle 10 Minuten. </li></ol> Teil 3: Ausführen der Durchflusszelle Experiment auf der BF1000-Workstation. <ol><li> Zunächst muss ein Satz ein automatisiertes Protokoll in der BIOFLUX Steuermodul. Für Kollagen I, sollte man die Einrichtung eines Protokolls zu 10dyn/cm 2 für 10 Minuten aus der Steckdose gut geführt. An dieser Stelle sollte man auch einrichten Datenerfassung Parameter mit dem BF1000-Workstation, einschließlich Kanal-Positionen, in FITC Wellenlänge und Zeitraffer-Informationen zu erfassen. Es wird empfohlen, 3 Felder der Ansicht mit einem 10-fach Objektiv pro Kanal alle 30 Sekunden über eine Dauer von 10 Minuten insgesamt zu erfassen. </li><li> Arbeiten schnell, Platz 500 ul der vorbereiteten Blut von jedem Zustand in getrennten Kanälen vorbereitet. Legen Sie keine Antikörper Kontrolle Blut in einen Kanal, der mit Kollagen und eine, die gerade gesperrt ist beschichtet ist. </li><li> Legen Sie die Schnittstelle auf der Platte. </li><li> Legen Sie die Platte auf der BF1000 Workstation Mikroskop. Clamp an der Schnittstelle. </li><li> Führen Platte Lasteinstellung. Auch bewegen Bühne, um ein gut mit Blut. Set FTIC Bilderfassung Parameter (Belichtungszeit, Verstärkung etc ...). </li><li> In der BIOFLUX montage-Software, starten Akquisition zu fließen und Akquisition gleichzeitig beginnen. </li><li> Die Platte aus BF1000-Workstation, der Platte nach den Richtlinien des Instituts zu entsorgen. </li></ol> Teil 4: Repräsentative Ergebnisse. Hier das Protokoll für die Adhäsion und Aggregation in mikrofluidischen Kanälen präsentiert wurde. Die Behandlung mit einem Thrombozytenaggregationshemmer, wurde anti-GPIIb/IIIa auch in das Protokoll aufgenommen. Mit Kollagen beschichtet mikrofluidischen Kanälen der BIOFLUX System, sollte man erwarten, dass aggressive Thrombusbildung im Laufe der Zeit sehen, mit der unbehandelten Kontrolle Blutprobe und keine Thrombenbildung auf der unbeschichteten Kanal. In einem kürzlich durchgeführten Experiment wurde die durchschnittliche Größe der Aggregate unter Kontrollbedingungen 2000μm 2. Abbildung 1. <img src="/files/ftp_upload/1644/1644fig1.jpg" alt="Abbildung 1" /> Aktiviert GPIIb / IIIa ist potenter Mediator der Thrombozyten-Thrombozyten-Interaktionen und Aggregation Stabilisierung 1. Die Haftung an Kollagen aktiviert den GPIIb / IIIa-Komplex zu dieser Antwort 2 entlocken. Nach der Inkubation mit anti-GPIIb/IIIa für 1 Stunde vor Scher-Exposition, sollte man erwarten, eine Abnahme in der Größe von Thromben sowie eine Abnahme der Häufigkeit von Thromben beobachten. Eine dosisabhängige Reaktion kann in der Regel bei 10dyn/cm 2 beobachtet werden. Abbildung 2. <img src="/files/ftp_upload/1644/1644fig2.jpg" alt="Abbildung 2" /> Die IC 50-Wert für diesen speziellen Inhibitor wurde 17Nm bei 10 dyn / cm 2. Die maximale Hemmung (für diese Spender) im Vergleich zu den nicht-Antikörper-Steuerung wurde um 11% auf 10 dyn / cm 2. Während die Methoden hier vorgestellt wurden unter Verwendung eines spezifischen extrazellulären Matrix-Protein und ein spezifischer Inhibitor der Thrombozytenaggregation, ist das Protokoll, erweiterbar auf andere Beschichtungen, andere Zelltypen und andere Inhibitoren für die Zelladhäsion Assays. Die wichtigsten Zutaten für den Erfolg solcher Experimente sind die Vermeidung von Blasen in der mikrofluidischen Kanälen, richtige Verdünnung aller Reagenzien in der richtigen Verdünnungsmittel und entsprechende Inkubationsbedingungen für alle Reagenzien.

<ol>
	<li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+complexity+of+platelet+aggregation.">The growing complexity of platelet aggregation.</a> Blood. 109, 5087-5095 (2007).</li><li>Nakamura, T., Kambayashi, J., Okuma, M., Tandon, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+GP+IIb-IIIa+complex+induced+by+platelet+adhesion+to+collagen+is+mediated+by+both+alpha2beta1+integrin+and+GP.">Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP.</a> VI. J Biol Chem. 274, 11897-11903 (1999).</li></ol>

Die Autoren sind Angestellte der Fluxion Biosciences.

Während die Methoden hier vorgestellt wurden unter Verwendung eines spezifischen extrazellulären Matrix-Protein und ein spezifischer Inhibitor der Thrombozytenaggregation, ist das Protokoll, erweiterbar auf andere Beschichtungen, andere Zelltypen und andere Inhibitoren für die Zelladhäsion Assays. Die wichtigsten Zutaten für den Erfolg solcher Experimente sind die Vermeidung von Blasen in der mikrofluidischen Kanälen, richtige Verdünnung aller Reagenzien in der richtigen Verdünnungsmittel und entsprechende Inkubationsbedingungen für alle Reagenzien.

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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>
		</thead>
		<tbody>
		
<tr>
<td>collagen I, Bovine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A1064401</td>
<td>other sources of collagen I can be used</td>
</tr>
<tr>
<td>PBS plus Ca/Mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin (BSA)</td>
<td>Reagent</td>
<td>EMD</td>
<td>EM-2930 Bottom of Form</td>
<td>From VWR</td>
</tr>
<tr>
<td>calcein AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C1430</td>
<td>Calcein AM from BD can also be used</td>
</tr>
<tr>
<td>BioFlux 1000 System</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>Contact Company</td>
<td>&nbsp;</td>
<tr>
<td>BioFlux Plate</td>
<td>Reagent</td>
<td>Fluxion Biosciences</td>
<td>900013</td>
<td>&nbsp;</td>
<tr>
<td>antiGPIIb/IIIa</td>
<td>Reagent</td>
<td>Abcam</td>
<td>ab33407</td>
<td>an alternative # is ab15021</td>
</tr>
<tr>
<td>ReoPro</td>
<td>Reagent</td>
<td>Eli Lilly</td>
<td>&nbsp;</td>
<td>by Rx only</td>
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platelet adhesion and aggregation under flow using microfluidic flow cells

Thrombozytenaggregation tritt als Reaktion auf eine Gefäßverletzung, wo der extrazellulären Matrix unter dem Endothel ausgesetzt war. Die Plättchenadhäsion Kaskade erfolgt in Gegenwart von Scherströmung, ein Faktor, nicht entfielen in konventionellen (statischen) Well-Platte-Assays. Dieser Artikel berichtet über eine Thrombozyten-Aggregation-Assay unter Verwendung eines mikrofluidischen Well-Platte-Format auf physiologische Scherströmung Bedingungen zu emulieren. Extrazelluläre Proteine, Kollagen I oder von Willebrand-Faktor innerhalb der mikrofluidischen Kanal mit aktiven Perfusion mit einer pneumatischen Pumpe hinterlegt. Die Matrix-Proteine ​​werden dann mit Puffer gewaschen und blockiert den mikrofluidischen Kanal für Thrombozyten-Interaktionen vorzubereiten. Vollblut mit Fluoreszenzfarbstoff markiert ist durch den Kanal bei verschiedenen Flussraten perfundiert, um die Thrombozyten-Aktivierung und Aggregation zu erreichen. Inhibitoren der Thrombozytenaggregation kann vor der Messzelle Experiment IC50 Dosis-Wirkungs-Daten zu generieren hinzugefügt werden.

Watch this Scientific Journal Video about Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells at JoVE.com

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Die Plättchenadhäsion Kaskade erfolgt in Gegenwart von Scherströmung, ein Faktor, nicht entfielen in konventionellen (statischen) Well-Platte-Assays. Dieser Artikel berichtet über eine Thrombozyten-Aggregation-Assay unter Verwendung eines mikrofluidischen Well-Platte-Format auf physiologische Scherströmung Bedingungen zu emulieren.

Adhäsion und Aggregation unter Fluss mit Mikrofluidik-Flow-Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

platelet-adhesion-aggregation-under-flow-using-microfluidic-flow

Universidad San Sebastian

Research

JoVE Journal

Biology

18.1K Aufrufe.  Fluxion Biosciences, Inc.. Die Plättchenadhäsion Kaskade erfolgt in Gegenwart von Scherströmung, ein Faktor, nicht entfielen in konventionellen (statischen) Well-Platte-Assays. Dieser Artikel berichtet über eine Thrombozyten-Aggregation-Assay unter Verwendung eines mikrofluidischen Well-Platte-Format auf physiologische Scherströmung Bedingungen zu emulieren.

Serie: Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

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.

Durchflusszytometrie Reinigung der Maus meiotischen Zellen

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 ...

Forschung

Biologie

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.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit 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 ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D-Bildgebung von Protein Aggregation in lebenden Zellen

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Mess-intrazellulären Ca<sup&gt; 2 +</sup&gt; Änderungen in menschlichen Spermien mit vier Techniken: Konventionelle Fluorometrie, Stopped Flußfluorometrie, Durchflusszytometrie und Single Cell Imaging

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Reduzierte Gravitationsbereich Hardware Demonstrationen eines Prototyps Miniaturisierte Durchflusszytometer und Companion Microfluidic Mischtechnik

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

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.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

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

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.

LED Thermo Flow - Die Kombination mit Optogenetik Flow Cytometry

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.

Analyse von Zellsuspensionen isoliert von festen Geweben von 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 ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Live-Zelle Bildgebung von Platelet Degranulation und Sekretion unter Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

Quantifizierung zellulärer Dichten und antigener Eigenschaften mittels Magnetschwebebahn

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...