تم دعم هذه الدراسة من خلال تطوير البرنامج الأكاديمي ذي الأولوية لمؤسسات التعليم العالي في جيانغسو (PAPD) ومشروع أبحاث إصلاح التدريس بجامعة الصين الصيدلانية (2019XJYB18).

1. إعداد وسط شبه صلب
<ol><li>أجار شبه صلب تقليدي
	<ol><li>تحضير الآجار شبه الصلب التقليدي وفقا لوصفة وسط اختبار حركية البكتيريا باستخدام المكونات الأساسية11. قم بإذابة 10 جم من التربتوز ، و 15 جم من كلوريد الصوديوم ، و 4 جم من أجار في كمية كافية من الماء المقطر ، واضبط الرقم الهيدروجيني على 7.2 ± 0.2 ، وشكل الحجم النهائي إلى 1000 مل.</li>
		<li>الأوتوكلاف أجار عند 121 درجة مئوية لمدة 20 دقيقة ، ووزعه في أنابيب اختبار 10 مل كوسط شبه صلب بارتفاع 3 سم.</li>
	</ol></li>
	<li>أجار شبه صلب تقليدي مع TTC
	<ol><li>بعد تعقيم الوسط شبه الصلب التقليدي ، قم بتبريده إلى 50 درجة مئوية ، وأضف 5 مل من محلول TTC المعقم 1٪ إلى 100 مل من الوسط ، واخلطه ، ووزعه في أنابيب اختبار 10 مل لتشكيل وسط شبه صلب بارتفاع 3 سم.</li>
	</ol></li>
</ol>2. السلالات البكتيرية
ملاحظة: تم عزل ثمانين سلالة من البيئة المائية وتحديدها باستخدام أداة آلية لتحديد البكتيريا (انظر جدول المواد)، بما في ذلك الإشريكية القولونية، والزائفة الزنجارية ( Salmonella spp.)، وضمة النيابة، والكلبسيلة الرئوية، وهيدروفيلا Aeromonas (الجدول 1). تم استخدام المكورات العنقودية الذهبية (انظر جدول المواد) كعنصر تحكم سلبي غير متحرك. تم استخدام الإشريكية القولونية والزائفة الزنجارية والسالمونيلا التيفية (انظر جدول المواد) كسلالات تحكم إيجابية.
<ol><li>تحديد السلالات البكتيرية الاختبارية لاستخدامها في تحليل الحركة.</li>
	<li>قم بتضمين عناصر تحكم سلبية غير متحركة وسلالات تحكم إيجابية متحركة.</li>
</ol>3. مراقبة الحركة البكتيرية المحسنة TTC
<ol><li>اختر مستعمرات مفردة من بكتيريا الاختبار من ألواح الآجار وقم بتلقيحها في الوسطين شبه الصلبين أعلاه (الخطوتان 1.1.2 و 1.2.1) عن طريق الثقب باستخدام إبر التلقيح.</li>
	<li>استزرع الأنابيب عند 37 درجة مئوية في الحاضنة لمدة 24-48 ساعة لمراقبة النتائج.</li>
	<li>راقب حالة النمو: وصف البكتيريا بأنها غير متحركة (-) إذا كان خط البزل أحمر فقط. قم بتمييز البكتيريا على أنها متحركة (+) إذا انتشر اللون الأحمر قليلا للخارج على طول خط البزل12.</li>
</ol>4. تأثير تركيزات الآجار المختلفة على الحركة البكتيرية
<ol><li>تحضير وسائط شبه صلبة تحتوي على 0.3٪ و 0.5٪ و 0.8٪ أجار وتلقيحها عن طريق ثقب ، كما هو موضح أعلاه. مراقبة النتائج بعد 24-48 ساعة من الحضانة.</li>
</ol>

<ol>
	<li>Jordan, E. O., Caldwell, M. E., Reiter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility.">Bacterial motility.</a> Journal of Bacteriology. 27 (2), 165 (1934).</li><li>Lai, S. L., Hou, H., Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility+and+its+role+during+initial+stage+of+pathogenesis.">Bacterial motility and its role during initial stage of pathogenesis.</a> Journal of Microbiology. 26 (5), 68-70 (2006).</li><li>Ding, S. S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+flagella-dependent+motility+and+biofilm+in+bacteria+-+A+review.">Relationship between flagella-dependent motility and biofilm in bacteria - A review.</a> Acta Microbiologica Sinica. 49 (4), 417-422 (2009).</li><li>Zeng, J., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+mechanism+of+bacterial+resistance+and+tolerance.">Recent advances in the mechanism of bacterial resistance and tolerance.</a> Chinese Journal of Antibiotics. 45 (2), 113-121 (2020).</li><li>Xu, M., Zhou, M. X., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+mechanism+of+bacterial+flagellum+motility,+adhesion+and+immune+escape.">Progress in the mechanism of bacterial flagellum motility, adhesion and immune escape.</a> Chinese Journal of Veterinary Science. 37 (2), 369-375 (2017).</li><li>Leboffe, M. J., Pierce, B. 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> Microbiology: laboratory theory and application. Third edition. , (2015).</li><li>Ball, R. J., Sellers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+motility+medium.">Improved motility medium.</a> Applied Microbiology. 14, 670-673 (1966).</li><li>An, S., Wu, J., Zhang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Pseudomonas+aeruginosa+biofilm+dispersal+by+a+cyclic-di-GMP+phosphodiesterase+with+a+putative+hypoxia-sensing+domain.">Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-di-GMP phosphodiesterase with a putative hypoxia-sensing domain.</a> Applied and Environmental Microbiology. 76 (24), 8160-8173 (2010).</li><li>Chouhan, O. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+site-directed+mutagenesis+at+the+GGEEF+domain+of+the+biofilm+forming+GGEEF+protein+from+Vibrio+cholerae.">Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.</a> AMB Express. 6 (1), 2 (2016).</li><li>McLaughlin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+colorimetric+microplate+test+of+phage+lysis+in+Salmonella+enterica.">Simple colorimetric microplate test of phage lysis in Salmonella enterica.</a> Journal of Microbiological Methods. 69 (2), 394-398 (2007).</li><li>Difco Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difco+manual:+Dehydrated+culture+media+and+reagents+for+microbiology.">Difco manual: Dehydrated culture media and reagents for microbiology.</a> Difco Laboratories. , (1984).</li><li>Tittsler, R. P., Sandholzer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility.">The use of semi-solid agar for the detection of bacterial motility.</a> Journal of Bacteriology. 31 (6), 575 (1936).</li><li>Qian, Y., Tian, X. Y., Zhang, S. Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explore+the+influencing+factors+of+bacterial+motility.">Explore the influencing factors of bacterial motility.</a> Health Care Today. 6, 50-51 (2018).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+Phytophthora+pathogens+deploy+effectors+to+interfere+with+bacterial+growth+and+motility.">Filamentous Phytophthora pathogens deploy effectors to interfere with bacterial growth and motility.</a> Frontiers in Microbiology. 11, 581511 (2020).</li><li>K&#252;hn, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments.">Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.</a> Nature Communication. 9 (1), 5369 (2018).</li><li>Mitchell, A. J., Wimpenny, J. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria.">The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.</a> Journal of Applied Microbiology. 83 (1), 76-84 (2010).</li><li>Xu, A., Zhang, M., Du, W., Wang, D., Ma, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+mechanism+for+how+sigma+factor+AlgT+and+transcriptional+regulator+AmrZ+inhibit+twitching+motility+in+Pseudomonas+aeruginosa.">A molecular mechanism for how sigma factor AlgT and transcriptional regulator AmrZ inhibit twitching motility in Pseudomonas aeruginosa.</a> Environmental Microbiology. 23 (2), 572-587 (2021).</li><li>Bartley, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+and+invasion+of+Neisseria+meningitidis+to+host+cells+is+related+to+surface+hydrophobicity,+bacterial+cell+size+and+capsule.">Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.</a> PLoS One. 8, 55798 (2013).</li></ol>

ليس لدى المؤلفين أي تضارب في المصالح للكشف عنه.

يتأثر اكتشاف الحركة البكتيرية بطريقة الوسط شبه الصلب بالعديد من العوامل13,14. يمكن أن تؤثر ظروف نمو البكتيريا ، مثل الأكسجين (الهوائية على سطح أجار ، غير الهوائية في الجزء السفلي من الأنبوب مع الوسط شبه الصلب) ، ودرجة الحموضة ، ودرجة الحرارة ، على صلاحية السوط...

تلعب الحركة البكتيرية دورا مهما في الإمراضية البكتيرية ، وتكوين الأغشية الحيوية ، ومقاومة الأدوية1. ترتبط الحركة البكتيرية ارتباطا وثيقا بالإمراضية وهي ضرورية للاستعمار البكتيري أثناء العدوى المبكرة للخلايا المضيفة2. يرتبط تكوين الأغشية الحيوية ارتباطا وثيقا بالحركة البكتيرية ، حيث تلتصق البكتيريا بسطح الوسائط الصلبة من خلال الحركة. لطالما اعتبرت الحركة البكتيرية مرتبطة بشكل إيجابي بتكوين الأغشية الحيوية. يمكن أن تؤدي درجة عالية من مقاومة البكتيريا للأدوية بسبب الأغشية الحيوية إلى عدوى مستمرة تشكل تهديدا لصحة الإنسان3،4،5. لذلك ، من المهم الكشف عن الحركة البكتيرية. يستخدم اختبار الحركة البكتيرية بشكل أساسي لفحص حركة أشكال مختلفة من البكتيريا في الحالة الحية ، والتي يمكن أن تحدد بشكل غير مباشر وجود أو عدم وجود سوط ، وبالتالي ، لها دور مهم في تحديد البكتيريا.
هناك طرق مباشرة وغير مباشرة للكشف عن الحركة البكتيرية6. نظرا لأن البكتيريا ذات السوط تظهر حركتها ، فمن الممكن اكتشاف ما إذا كانت البكتيريا متحركة بشكل غير مباشر عن طريق اكتشاف وجود أو عدم وجود سوط. على سبيل المثال ، من الممكن اكتشاف الحركة بشكل غير مباشر عن طريق المجهر الإلكتروني وتلطيخ السوط للإشارة إلى أن البكتيريا متحركة. من الممكن أيضا اكتشافه بالطرق المباشرة ، مثل طرق إسقاط التعليق والبزل شبه الصلب. 
طريقة البزل شبه الصلبة المستخدمة عادة في مختبرات الأحياء الدقيقة الجامعية للكشف عن الحركة البكتيرية تقوم بتلقيح البكتيريا في ثقب في وسط أجار شبه صلب يحتوي على 0.4-0.8٪ أجار ، وفقا لاتجاه نمو البكتيريا. إذا نمت البكتيريا على طول خط البزل لتنتشر حولها ، تظهر آثار نمو تشبه السحابة (تشبه الفرشاة) ، مما يشير إلى وجود سوط ، وبالتالي الحركة. إذا لم تكن هناك آثار لنمو خط البزل ، فإن البكتيريا ليست جلدية ولا متحركة. 
ومع ذلك ، فإن هذه الطريقة لها عيوبها: البكتيريا عديمة اللون وشفافة ، ويتأثر النشاط السوطي بالخصائص الفسيولوجية للبكتيريا الحية وعوامل أخرى ، وتركيز أجار والقطر الصغير لأنبوب الاختبار. علاوة على ذلك ، فإن البكتيريا الهوائية مناسبة فقط للنمو على سطح أجار ، مما يؤثر على مراقبة الحركة البكتيرية. ومن ثم ، لتحسين هذه التجربة ، تمت إضافة 2،3،5-ثلاثي فينيل تترازوليوم كلوريد (TTC) (عديم اللون) إلى الوسط لإنشاء طريقة أكثر موثوقية وبديهية لتحديد حركية البكتيريا من طريقة البزل المباشر الحالية باستخدام نازعة الهيدروجين داخل الخلايا لتحفيز تكوين منتج أحمر من TTC7،8،9،10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>ATCC25922</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>ATCC</td>
			<td>ATCC27853</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>ATCC25923</td>
			<td>Negative nonmotile control</td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>&#160;OXOID</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Sigma</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>VITEK 2 automated microbial identification system</td>
			<td>Bio M&#233;rieux</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing bacterial motility based on a color reaction

الحركة البكتيرية أمر بالغ الأهمية للإمراضية البكتيرية ، وتكوين الأغشية الحيوية ، ومقاومة الأدوية. الحركة البكتيرية أمر بالغ الأهمية لغزو و / أو نشر العديد من الأنواع المسببة للأمراض. لذلك ، من المهم الكشف عن الحركة البكتيرية. يمكن أن تؤثر ظروف نمو البكتيريا ، مثل الأكسجين ودرجة الحموضة ودرجة الحرارة ، على نمو البكتيريا والتعبير عن السوط البكتيري. هذا يمكن أن يؤدي إلى انخفاض الحركة أو حتى فقدان الحركة ، مما يؤدي إلى تقييم غير دقيق للحركة البكتيرية. استنادا إلى تفاعل اللون لكلوريد 2،3،5-ثلاثي فينيل تيترازوليوم (TTC) بواسطة نازعة الهيدروجين داخل الخلايا للبكتيريا الحية ، تمت إضافة TTC إلى أجار شبه صلب تقليدي للكشف عن الحركة البكتيرية. أظهرت النتائج أن طريقة أجار TTC شبه الصلبة للكشف عن الحركة البكتيرية بسيطة وسهلة التشغيل ولا تتضمن أدوات كبيرة ومكلفة. كما أظهرت النتائج أن أعلى حركة لوحظت في وسط شبه صلب محضر بنسبة 0.3٪ أجار. بالمقارنة مع الوسط شبه الصلب التقليدي ، فإن النتائج أسهل في التقييم وأكثر دقة.

تمت مقارنة كل من السلالات القياسية والسلالات المعزولة للكشف عن الحركة ، والنتائج موضحة في الجدول 1. بسبب عدم وجود سوط ، نمت المكورات العنقودية الذهبية والكلبسيلة الرئوية فقط على طول الخط الملقح على كل من الوسائط شبه الصلبة التقليدية و TTC. في المقابل ، أظهرت الزائفة الز...

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

هنا ، نقدم بروتوكولا للكشف عن الحركة البكتيرية بناء على تفاعل اللون. المزايا الرئيسية لهذه الطريقة هي أنها سهلة التقييم وأكثر دقة ، ولا تتطلب معدات متخصصة.

تصور الحركة البكتيرية بناء على تفاعل اللون

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

visualizing-bacterial-motility-based-on-a-color-reaction

Research

JoVE Journal

Biology

3.7K Views.  China Pharmaceutical University. هنا ، نقدم بروتوكولا للكشف عن الحركة البكتيرية بناء على تفاعل اللون. المزايا الرئيسية لهذه الطريقة هي أنها سهلة التقييم وأكثر دقة ، ولا تتطلب معدات متخصصة.

Video: Visualizing Bacterial Motility Based on a Color Reaction

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

تقييم العصبية على الحركة باستخدام الخلايا الجذعية جل المستندة Agarose الأجهزة ميكروفلويديك

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

تصور تكرار الحمض النووي جزيء واحدة مع مضان المجهري

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

تصور التعريب في الحمض النووي الريبي القيطم البويضات

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

تصور الإلتقام بالكلاذرين بوساطة من G مستقبلات البروتين يقترن قرار واحد في الحدث عبر الميكروسكوب TIRF

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

التحضير والتصوير، والكمي لالسطحية الجرثومي على الحركة فحوصات

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

قياس وتحليل حمض خارج الخلية الإنتاج لتحديد معدل حال السكر

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-Galactosidase القائم على فحص الفحص لتحديد الأدوية Senotherapeutic

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

طريقة فيفو السابقة لتقييم الحركة التلقائية للجهاز التناسلي للماوس وخوارزمية تتبع حركة الرحم المستندة إلى MATLAB لتحليل البيانات

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

نموذج يستند إلى كبسولة لمراحل القراد الثابت غير ناضجة الإصابة على الفئران المختبرية

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

تصور وقياس الأضرار التي لحقت بالحمض النووي الخاصة بالموقع القائم على الإندونوكليز

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...