وقد تم تمويل هذا العمل عن طريق منحة من المعاهد القومية للصحة (GM070736) لحاء Fölsch. كان مدعوما من قبل SF آنغ A STAR * الجائزة منحة دراسية عليا ، وكان مدعوما من قبل RS كانغ أساس الخلوية والجزيئية لبرنامج التدريب على الأمراض (GM8061)

<ol>
	<li>Mellman, I., Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 9 (11), 833-833 (2008).</li><li>Rodriguez-Boulan, E., Kreitzer, G., Musch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 6 (3), 233-233 (2005).</li><li>Ang, A. L., F&#246;lsch, H., Koivisto, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 163 (2), 339-339 (2003).</li><li>Kroschewski, R., Hall, A., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 1 (1), 8-8 (1999).</li><li>Moskalenko, S., Henry, D. O., Rosse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 4 (1), 66-66 (2002).</li><li>Schuck, S., Gerl, M. J., Ang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traffic (Copenhagen, Denmark). 8 (1), 47-47 (2007).</li><li>Nokes, R. L., Fields, I. C., Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 182 (5), 845-845 (2008).</li><li>Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cell.">Molecular cell.</a> 12, 1064-1064 (2003).</li><li>Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science (New York, N.Y. 279 (5350), 509-509 (1998).</li><li>Scales, S. J., Pepperkok, R., Kreis, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 90 (6), 1137-1137 (1997).</li><li>Keller, P., Toomre, D., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 3 (2), 140-140 (2001).</li></ol>

الإعلان عن أي تضارب في المصالح.

الخطوات الأكثر أهمية بالنسبة لتجربة microinjection الناجحة هي جودة ونقاء من الحمض النووي والتقاطب من الخلايا الخاصة بك. بدون خلايا الاستقطاب ، وسوف تسيطر بالفعل الخاص حقن وmistargeted VSVG ولا يمكن استخدامها لتكون التجربة. إذا كان الحمض النووي هو من نوعية رديئة ، قد دنا تسد إبرة ا?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> Axiovert 200 مجهر مع مرحلة ساخنة </td><td> شركة كارل زايس </td><td> عرف النظام </td></tr><tr><td> Injectman micromanipulator NI2 Femtojet </td><td> إيبندورف </td><td> عرف النظام </td></tr><tr><td> Femtotips الثاني (الإبر Microinjection) </td><td> إيبندورف </td><td> 930000043 </td></tr><tr><td> نصائح Microloader </td><td> إيبندورف </td><td> 930001007 </td></tr><tr><td> 12 ملم واضحة transwell يدعم مرشح </td><td> كورنينج Costar </td><td> 3460 </td></tr><tr><td> الذيفان الداخلي خالية عدة maxiprep البلازميد </td><td> سيغما الدريخ </td><td> NA0400 </td></tr></tbody></table>

analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells

الخلايا الظهارية استقطاب غشاء البلازما الخاصة بهم في كيميائيا ومتميزة وظيفيا المجالات قمية وbasolateral حيث المجال قمية يواجه &#39;الحرة&#39; السطوح والغشاء basolateral على اتصال مع الركيزة والخلايا المجاورة. ويفصل غشاء كلا المجالات التي منعطفات ضيقة ، والتي تشكل حاجزا نشرها. ويمكن تلخيصها قمية - basolateral بنجاح في ثقافة الاستقطاب عند المصنف الخلايا الظهارية مثل الكلى الناب مدين - داربي (MDCK) في الخلايا على مرشحات عالية الكثافة والبولي ومثقف لعدة أيام 1 2. وينظم إنشاء وصيانة قطبية الخلية من قبل مجموعة من GTPases صغير من الفصيلة مثل رأس رالا ، Cdc42 ، Rab8 ، وRab10 Rab13 3 4 5 6 7. مثل كل GTPases هذه البروتينات دورة الناتج المحلي الإجمالي بين دولة محددة نشط وفعال دولة GTP محدد. طفرات معينة في مناطق الربط النوكليوتيدات تتداخل مع هذه الدراجات 8. على سبيل المثال ، يتم تأمين دائم Rab13T22N في شكل الناتج المحلي الإجمالي ، وبالتالي يطلق عليها اسم &quot;المهيمن السلبية&quot; ، في حين لم تعد قادرة على Rab13Q67L يتحلل GTP ومؤمن وبالتالي في حالة &quot;نشطة المهيمنة&#39; 7. لتحليل وظيفتها في الخلايا عادة يتم التعبير عن كل من الأليلات السائدة السلبية والنشطة GTPases المهيمن على مستويات عالية للتدخل في وظيفة من البروتينات الذاتية 9. طريقة أنيقة لتحقيق مستويات عالية من overexpression في فترة قصيرة من الوقت هي لتعريف البلازميدات ترميز البروتينات ذات الصلة مباشرة في نوى خلايا الاستقطاب نمت على تصفية تدعم استخدام تقنية microinjection. غالبا ما يتم الجمع بين هذا مع الحقن المشترك للالبلازميدات مراسل تكود مستقبلات غشاء البلازما التي يتم فرزها على وجه التحديد إلى المجال أو قمية basolateral. وكثيرا ما تستخدم لشحن البضائع تحليل الفرز إلى المجال basolateral هو أليل درجة الحرارة حساسة من فيروس التهاب الفم الحويصلي بروتين سكري (VSVGts045) 10. لا يمكن لهذا البروتين بشكل صحيح في حظيرة 39 درجة مئوية ، وبالتالي فسيتم الاحتفاظ بها في شبكية هيولي باطني (ER) ، في حين يتم تجميع البروتين التنظيمية للمصلحة في العصارة الخلوية. التحول إلى 31 درجة مئوية وسوف يسمح لأمثال ثم VSVGts045 بشكل صحيح ، وترك لائحة والسفر إلى غشاء البلازما 11. عادة ما يتم تنفيذ هذه المطاردة في حضور سيكلوهيكسيميد لمنع المزيد من تخليق البروتين يؤدي إلى نتائج أكثر نظافة. نحن هنا تصف بالتفصيل إجراءات microinjecting البلازميدات الى خلايا الاستقطاب وحضانات التحولات اللاحقة بما في ذلك درجة الحرارة التي تسمح لتحليل شامل من البروتينات التنظيمية المشاركة في الفرز basolateral.

Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

هذه المقالة تفاصيل الإجراءات المتعلقة overexpression وتحليل GTPases صغيرة في الخلايا الظهارية الاستقطاب باستخدام تقنية microinjection.

تحليل وظيفة GTPases الصغيرة التي Microinjection من البلازميدات إلى الخلايا الظهارية المستقطب

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

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Biology

11.3K Views.  Northwestern University. هذه المقالة تفاصيل الإجراءات المتعلقة overexpression وتحليل GTPases صغيرة في الخلايا الظهارية الاستقطاب باستخدام تقنية microinjection.

Video: Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjection الجنين اسماك الزرد إلى تحليل وظائف الجينات

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

الخلايا الظهارية الثديية شكل الماوس Mammospheres خلال التمايز المولد للبن

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Intranuclear Microinjection من الحمض النووي في الخلايا العصبية فصل الثدييات الكبار

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

تحليل حركية مرنا الصادرات النووية في خلايا الثدييات التي Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

تفعيل موت الخلايا المبرمج من قبل Microinjection هيولي من السيتوكروم ج</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

لونين عزل من قبل تنقية الحمض النووي الريبي (السقسقة)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

زراعة خلايا الإنسان الظهارية الأنف في واجهة الهواء السائل

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

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

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

قياس الكالسيوم عصاري خلوي في الخلايا الظهارية الكلوية بواسطة التدفق الخلوي

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

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.