The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

<html> 1. הכנת תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לשמור על תרבות של קו תא אפיתל הרתי (IT76M1) או תא להיבדק באינקובטור (37 ° C / 5% CO 2). </li><li style=";text-align:right;direction:rtl"> שוטפים את התאים עם PBS 1x (לחזור פריט זה 3x). </li><li style=";text-align:right;direction:rtl"> להוסיף טריפסין על התאים למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף בינוני (פעמים מהיקף טריפסין הוסיף בסעיף 1.3) עם 10% FBS (בסרום שור עוברי) אל התאים עם טריפסין ו צנטריפוגות (800 XG במשך 5 דקות). </li><li style=";text-align:right;direction:rtl"> ספירת התאים ב hemocytometer. </li><li style=";text-align:right;direction:rtl"> התאם את הצפיפות של תאים בהתאם לסוג התא כמו התאים צריכים להיות בקשר הדוק עם שני כדי לאפשר צימוד. הערה: במקרה שלנו, השתמשנו 3 x 10 5 תאים לכל 35 מ&quot;מ צלחת פטרי. </li></ol> 2. הכנת Micropipette <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> משוך את micropipette כמפורט מתוך micropipette נימי זכוכית (1.5 מ&quot;מ קוטר) על סופי 0.2 מיקרומטר של קוטר כדי להשיג התנגדות סופית של כ -30 MΩf &quot;&gt; 17, 18. הערה: לחלופין, טפטפות הזרקה ניתן לרכוש. ההתנגדות תלוי בגודל התא, כמו למשל microelectrode עמידות גבוהה יותר יהיה צורך עבור תאי acinar הלבלב, למשל (100-150 MΩ) 19. בעיה נפוצה שעלולה להתרחש היא ממטרים של פתרון צהוב לוציפר אז מה שיכול להכשיל את micropipette והן עשויות לחייב סינון או צנטריפוגה מוקדמת. לפני ההזרקה, micropipette יש לנתח תחת מיקרוסקופ כדי לזהות אם קיימת חסימה או כל סוג של הפרעה 13. את micropipette יכול להיבדק על ידי הזרקת LY עם קצה micropipette בתוך תמיסת מלח. </li></ol> 3. בדיקת Micropipette <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את הפתרון צהוב לוציפר (5%) ב -150 mmol / L LiCI ולטעון את micropipette באמצעות מזרק או למלא את משבצות (מכניס אותו פתרון LY). </li><li style=";text-align:right;direction:rtl"> מניחים את micropipeטטי על צלחת פטרי 35 מ&quot;מ עם תאי IT76M1 בתחנת עבודת microinjection ו להטביע את קצה micropipette הזכוכית לתוך המדיום הסלולרי. דגש על micropipette ולבצע בדיקה זורם לצבוע ידי החלת דופק. </li></ol> 4. תא בודד לוציפר צהוב Microinjection <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פוקוס מיקרוסקופ ממש מעל שכבת התאים באמצעות קטיון fi Magni גבוהה (40X), ואז להנמיך את פיפטה לאט אל התאים באמצעות micromanipulator. </li><li style=";text-align:right;direction:rtl"> לנקב את תא המטרה כאשר הקצה הוא קרוב עד כדי נגיעת קרום התא, ולהחיל דופק hyperpolarizing קטן להציג את LY לתוך התא. המתח להחיל יהיה תלוי מטען נטו של צבע כדי להיות מוזרק. לחילופין, ישנם צבעים אחרים יכולים לשמש עם הטכניקה הזו כפי שמוצג בטבלה 1. הערה: באופן עקרוני כל צבע הידרופילי עם MW פחות 1KDa יוכל לשמש. עם זאת, את קצב ההעברה יכול להשתנות על פי המשקל hydrophilicity. בנוסף, uהעברת Nspecific של צבע המשמש חייבת להיות מוערכת. </li><li style=";text-align:right;direction:rtl"> צלמו תמונות תא 3 דקות לאחר הזרקת צבע או לעשות סרט קטן עם מיקרוסקופיה זמן לשגות (30 fps). הערה: גישה דומה ניתן היה לראות היטומי ואח &#39;(2015) 20. כדי למנוע תקשורת באמצעות גשרים בין תאית (מיטוזה שלמה) חברת dextran שיתוף הזרקת rhodamin (מ 2 עד 10 KDA), אשר אינו עוברת דרך צומת פער אבל עוברת דרך גשרים בין תאית וסוגים מסוימים של nanotubules המומלץ כפי שמוצג באיור 2 . </li></ol>

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The authors have no conflicts of interest.

כדי לאמת את הנוכחות של צומת הפער בין תאית פונקציונלי, להשתמש באמצעים, אשר קרום חדיר, למרות חדיר ידי ערוצי אינטר נדרשים 16. והעמסה, צבע פלואורסצנטי הראשון להבחין תא אל תא צימוד 22, הוא חדיר בין ממברנות הלא junctional 3 ו הוחלפה ולכן על ידי ...

צמתים הפער הם ערוצי אינטר המאפשרים אינטרקום בין תאים שכנים 1. תקשורת זו המחברת בין שני תאים או יותר השכנות, שבהן כל אחד תורם עם connexon או hemichannel כדי ליצור את ערוץ אינטר. בתאי יונקים, את connexon נוצר על ידי שישה connexins, מונומרים עם ארבעה תחומים הטרנסממברני ו- C ו- N מסוף בתוך הציטופלסמה 2. צמתים הפער לא רק לאפשר מעבר של יונים, שליחים משניים מטבוליטים קטנים, אלא גם לתרום הרבה צורות של תקשורת סלולרית בתהליכים פיזיולוגיים רבים, כגון העברה סינפטית, התכווצות הלב, צמיחת תאים ו -3 בידול, 4, 5, 6, 7, 8. בנוסף צומת פער נקשרומחלות רבות כולל סרטן 9, 10, ניוון שרירים 11, כמה מחלות גנטיות demyelinating 12 מחלות. סוג זה של crosstalk בין תאית יכול להיות מוערך על ידי מספר שיטות 13, 14, 15, 16. במאמר זה, אנו מראים כיצד לבצע microinjection מתחיל מתא בודד הצהוב לוציפר לדמיין תקשורת סלולרית באמצעות-צומת פער בתאים חיים. אנו דנים כיצד להכין את התאים ואת micropipette, את השימוש של micromanipulator והזרמת לצבוע צהוב לוציפר בקו תא אפיתל הרתי. בדרך כלל, הליך ניסיוני זה יכול להיות מנותח על ידי הממוצע של תאי מחוברי התא טעון עם צבע. בנוסף, שיטה זו יכולה לשמש עם צבעי ניאון אחרים עם משקל מולקולרי מתחת הפערצומת חתוכים המהווה כ -1,000 Daltons.

<table border="0" cellpadding="0" cellspacing="0" width="1228">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lucifer yellow</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L0259</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lithium Chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L4408</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS tablets</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:137px;">P4417</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">R4130</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Bovine fetal serum</td>
			<td style="width:71px;">Cultilab</td>
			<td style="width:137px;"></td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Trypsin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">T4799</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:137px;">TE-2000</td>
			<td>For microinjection experiments, one needs an inverted fluorescence microscope and &#64257;lters for &#64258;uorescent microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibration-insulated table&#160;</td>
			<td style="width:71px;">Newport</td>
			<td style="width:137px;">VH3036W-OPT</td>
			<td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:137px;">MMO-203</td>
			<td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Current Generator&#160;</td>
			<td style="width:71px;">Digitimer</td>
			<td style="width:137px;">DS2</td>
			<td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&#160;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

single-cell microinjection for cell communication analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

תא אפיתל הרתי קו IT-76MI שמש להעריך צימוד לצבוע ידי צומת פער כמו תאים אלה תוארו להביע צומת פער פונקציונליים נוצרו על ידי connexin 43 21. איור 1 מציג את הזריקה של לוציפר צהוב כשהם מיושמים בתא אחד מתחת קצה פיפטה. לאחר כמה דקות, תאים המחוברים להיות ניא...

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis

אנו מתארים כאן כיצד לבצע microinjection מתחיל מתא בודד הצהוב לוציפר לדמיין תקשורת סלולרית באמצעות-צומת פער בתאים חיים, ולספק כמה עצות שימושיות. אנו מצפים כי מסמך זה יעזור לכולם להעריך את מידת צימוד הסלולר בשל צומת פער פונקציונליים. הכל המתואר כאן יכול להיות, באופן עקרוני, מותאמי צבעי ניאון אחרים עם משקל מולקולרי להלן 1,000 Daltons.

Microinjection מתא בודד לניתוח תקשורת נייד

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

single-cell-microinjection-for-cell-communication-analysis

Research

JoVE Journal

Biology

11.2K Views.  Oswaldo Cruz Foundation.  אנו מתארים כאן כיצד לבצע microinjection מתחיל מתא בודד הצהוב לוציפר לדמיין תקשורת סלולרית באמצעות-צומת פער בתאים חיים, ולספק כמה עצות שימושיות. אנו מצפים כי מסמך זה יעזור לכולם להעריך את מידת צימוד הסלולר בשל צומת פער פונקציונליים. הכל המתואר כאן ...

Video: Single-cell Microinjection for Cell Communication Analysis

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

תא יחיד transfection בעוברים צ&#39;יק

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

תא בודד הגורל מיפוי של דג הזברה

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

פרופיל תא יחיד תעתיק של cardiomyocytes עכבר למבוגרים

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

ניתוח של שעתוק גנים מהתא הבודד על ידי RNA פלורסנט<em&gt; באתר</em&gt; הכלאה (דגים)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

ניתוח Microfluidic תא בודד של Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

ניתוח של תפקוד תאי בטא באמצעות הדמיה הסידן בתא יחיד ברזולוציה איונים דג זברה

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

הדמיה תלת-ממדית ברזולוציה חד-תאית של אורגנואידים שלמים

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

זיהוי בזמן אמת של ייצור מיני חמצן תגובתי בתגובה חיסונית באורז עם בדיקת כימילומינסנציה

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down to the level of a single cell. Experiments using this method have achieved quantifying over 3,000 proteins across 1,500 single mammalian cells (500-1,000 proteins per cell) in 10 days of mass spectrometer instrument time. SCoPE2 leverages a freeze-heat cycle for cell lysis, obviating the need for clean-up of single cells and consequently reducing sample losses, while expediting sample preparation and simplifying its automation. Additionally, the method uses an isobaric carrier, which aids protein identification and reduces sample losses.
This video protocol provides detailed guidance to enable the adoption of automated single-cell protein analysis using only equipment and reagents that are widely accessible. We demonstrate critical steps in the procedure of preparing single cells for proteomic analysis, from harvesting up to injection to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, viewers are guided through the principles of experimental design with the isobaric carrier, quality control for both isobaric carrier and single-cell preparations, and representative results with a discussion of limitations of the approach.

In this protocol, we describe how to prepare mammalian cells for single-cell proteomics analysis via mass spectrometry using commercially available reagents and equipment, with options for both manual and automatic pipetting.

הכנה לפרוטאומיקה חד-תאית לניתוח ספקטרומטריית מסה באמצעות ליזיס הקפאת חום ונשא איזוברי

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the ...