الحلول <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحل بين الخلايا لخلايا العقدة (مم) : 100 K - غلوكونات ، 8 بوكل ، 1 MgCl 2 ، 1 EGTA ، 10 HEPES ، 4 ATP ، GTP 0.5 و 90 ميكرومتر Sulforhodamine باء (لتلوين) ، بعد تعديلها لدرجة الحموضة = 7.4 مع KOH </li><li style=";text-align:right;direction:rtl"> الحل بين الخلايا لخلايا القطبين (مم) : 90 K - غلوكونات ، 8 بوكل ، 1 MgCl 2 ، 10 BAPTA ، 10 HEPES ، 4 ATP ، GTP 0.5 و 90 ميكرومتر Sulforhodamine باء (لتلوين) ، بعد تعديلها لدرجة الحموضة = 7.4 مع KOH </li><li style=";text-align:right;direction:rtl"> محلول رينغر خارج الخلية (مم) : 112 كلوريد الصوديوم ، الجلوكوز 5 ، 5 HEPES ، 2 بوكل ، 2 CaCl 2 ، 1 2 MgCl ، بعد تعديلها لدرجة الحموضة = 7.75 مع هيدروكسيد الصوديوم. الأوكسجين من الحل وليس من الضروري. </li></ul> إعداد غرف تسجيل لدينا غرفة تسجيل تتكون من شريحة المجهر بمادة لاصقة جيدا في السيليكون الذي يمنع قطع صغيرة أخرى لاصقة وضعت بشكل جيد لتوفير الاستقرار لشرائح الأنسجة. إعداد شرائح الشبكية لتجنب التبييض من المستقبلات الضوئية ، ويتم تشريح في ضوء الأشعة تحت الحمراء أو خافت الضوء الأحمر. إذا كان أحد يرغب في تسجيل الاستجابات من المسار قضيب ، يجب استخدام الضوء الوحيد IR (الأشعة تحت الحمراء المجهر وتشريح أي ضوء الغرفة ، وينبغي استخدام نظارات الأشعة تحت الحمراء الإضافية لإجراءات لم تفعل تحت المجهر). يمكن إذا كان أحد يهتم أساسا في ضوء تسجيل الاستجابات المخروط ، الضوء الخافت الغرفة الحمراء أو ضوء الفلاش الحمراء يمكن استخدامها. المجهر ، ومع ذلك ، ينبغي أن يكون لا يزال oculars الأشعة تحت الحمراء. إذا الإدخال مبصرة لا يمت بصلة لهذه التجربة ، يمكن القيام بإجراءات في ضوء الغرفة العادي باستخدام المجهر العادية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير الشرائح المجهر two مع سيليكون لاصقة أيضا : إنشاء اثنين من نطاقات متوازية من الشحوم السيليكون (حوالي 0.5 سم بعيدا) على كل الشرائح ووضع قطعة مستطيلة من الورق تصفية على جانب واحد من الشرائح ، سواء معبر العصابات الشحوم (دفع التصفية مع جزء من ورقة شفرة حلاقة بحيث يتم المرفقة جيدا إلى الشريحة) </li><li style=";text-align:right;direction:rtl"> تضحية السمندل المنشأة وفقا لبروتوكولات : يتم وضع الحيوانات في حمام الثلج لمدة 30 دقيقة ، ثم مقطوعة الرأس وضعف pithed. </li><li style=";text-align:right;direction:rtl"> مبلل إزالة العين من الرأس السمندل ووضعه تحت مجهر تشريح على قطعة من المناديل الورقية مع الحل الجليد الباردة رينغر </li><li style=";text-align:right;direction:rtl"> إزالة النسيج الضام من العين </li><li style=";text-align:right;direction:rtl"> استخدام شفرة حلاقة لإجراء قطع صغير في القرنية </li><li style=";text-align:right;direction:rtl"> عقد قطع القرنية مع ملقط حين إزالته بالمقص على طول حرصه على العين </li><li style=";text-align:right;direction:rtl"> إزالة العدسة مع ملقط </li><li style=";text-align:right;direction:rtl"> عقد القزحية مع ملقط وقطع على طول مشرشر أورا لإزالة قزحية </li><li style=";text-align:right;direction:rtl"> قطع فنجان العين في اثنين قطع مستطيلة </li><li style=";text-align:right;direction:rtl"> التقط واحدة من القطع الصلبة وضعه الجانب حتى على ورقة الترشيح (للتأكد من أصل قطعة تتسطح في حين تضغط عليه بعناية فائقة) </li><li style=";text-align:right;direction:rtl"> رفع بلطف الصلبة قبالة شبكية العين ، والتي ينبغي الآن على الورقة المرفقة المدون </li><li style=";text-align:right;direction:rtl"> إضافة الحل بسرعة الجليد الباردة رينغر إلى البئر </li><li style=";text-align:right;direction:rtl"> تكرار نفس القطعة الثانية </li><li style=";text-align:right;direction:rtl"> استخدام شفرة حلاقة وضعت في تقطيع اللحم والعرف لخفض 200 300μm شرائح واسعة. </li><li style=";text-align:right;direction:rtl"> استخدام زوج من ملقط لالتقاط الشرائح على جانب ورقة الترشيح ، تسليمهم حتى طبقات الشبكية تصبح مرئية ، ووضعها على نطاقات الشحوم فراغ </li><li style=";text-align:right;direction:rtl"> اختيار شريحة التي تبدو سليمة ويبين الفرق أفقي يشير إلى طبقات الشبكية (طبقات في بعض الأحيان من الصعب أن نرى تحت مجهر التشريح والتكبير ليست عالية بما فيه الكفاية ، ولكن إذا كان يمكن للمرء أن يتعرف على بعض طبقات شريحة عادة جيدة بما فيه الكفاية. القرار النهائي بشأن ما إذا كان استخدام شريحة ، ولكن ، مرة واحدة وينظر تحت المجهر تستقيم). نقله إلى غرفة تسجيل الشريحة من خلال بناء جسر بين الشرائح رينغر two </li><li style=";text-align:right;direction:rtl"> تأكد من دفع ورقة الترشيح ضد كتل المطاط في غرفة تسجيل شريحة بحيث لا يميل (خلايا العقدة مبصرات وينبغي أن يكون في التركيز في نفس الوقت) </li></ol> وضع الأنسجة في تلاعب تسجيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم وضع الشريحة تسجيل تحت المجهر وperfused مع حلول رينغر من خلال الجاذبية التنفسي نضح </li><li style=";text-align:right;direction:rtl"> إعداد تصور هو مع المجهر تستقيم مع الهدف الماء الغمر 10x و40X. </li><li style=";text-align:right;direction:rtl"> مرة أخرى ، ونحن فقط استخدام ضوء الأشعة تحت الحمراء لمنع تبييض للمبصرات. ويرد كاميرا الأشعة تحت الحمراء إلى جانب منفذ المجهر ، وهذا مرتبط إلى شاشة التلفزيون. </li></ol> التحفيز البصري مبرمجة المحفزات البصرية في Matlab باستخدام Psychtoolbox ويتوقع على شبكية العين عن طريق ميناء رأس مجهر الفحص المجهري باستخدام محقن صورة (MBF العلوم الحيوية). A بتات + + يستخدم معالج الفيديو الرقمي (كامبريدج لأبحاث الأنظمة) للحصول على نطاق والإنارة و14bit synchronize عرض الحوافز مع الحصول على البيانات. والمحفزات البصرية المستخدمة في هذا الفيديو وأشرطة مشرق أو داكنة (العرض = 460μm) على النقيض من 100 ٪ ، والتي قدمت لمدة ثانيتين على خلفية موحدة ثابتة (الإنارة = 8 * 10 4 الفوتونات / ميكرون / ثانية ، الحجم : 1.84 س 1.38mm). الكهربية <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم سحبها أقطاب التصحيح من الزجاج البورسليكات (OD :. 1.5mm ، ID 84mm) مع مجتذب micropipette. للخلايا العقدة ، المقاومة هي ما بين 2 و 4 MOhms. للخلايا بين القطبين ، وتستخدم المقاومة العالي ما بين 4 و 8 MOhms. </li><li style=";text-align:right;direction:rtl"> تجرى التسجيلات المشبك الجهد باستخدام 700A Multiclamp (الأجهزة الجزيئية) ، ومكبر للصوت المختبر البرامج المبرمجة في LABVIEW الداخلية. </li><li style=";text-align:right;direction:rtl"> وperfused المخدرات من خلال نظام الارواء 8 قنوات. يوضع القطب microperfusion قريبة من شريحة (النسيج يجب أن تتحرك بشكل واضح عند تشغيل نضح وخارجها ، ولكن يحتاج المرء أن يكون حذرا من ان الضغوط ليست قوية جدا بحيث يمكن أن تصبح شريحة منفصلة عن ورقة الترشيح). وينبغي أن يكون خلال التجارب microperfusion تحول باستمرار على (حل perfusing السيطرة إذا ليست هناك حاجة المخدرات) بحيث لا يتم تسجيل منزعجة من الحصول على تبديل نضح وخارجها. </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

الفوائد : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جميع أنواع الخلايا متاحة </li><li style=";text-align:right;direction:rtl"> من السهل تحديد من أنواع الخلايا ، وبخاصة إذا تم إضافة صبغة الفلورسنت إلى الحل الكهربائي </li><li style=";text-align:right;direction:rtl"> ويمكن الوصول بسهولة وكلاء الدوائية الخلاي?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Multiclamp 700A</td>
<td>Patch Clamp Amplifier</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SZX9</td>
<td>Dissection Micropscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BX50WI</td>
<td>Upright Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td>
</tr>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lucivid</td>
<td>Microscope image injector</td>
<td>Mbf bioscience</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>8-channel perfusion system</td>
<td>Microperfusion</td>
<td>Parker Hannfin corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Digital Video Processor</td>
<td>Cambridge Research Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Oculars</td>
<td>Other</td>
<td>ITT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive silicone wells</td>
<td>Other</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>20mm diameter, 1.0mm deep</td>
</tr>
<tr>
<td>Membrane Filters</td>
<td>Filter</td>
<td>Milipore</td>
<td>HAWPO1300</td>
<td>.45&#181;m HA</td>
</tr>
<tr>
<td>Borosilicate Glass Capillaries</td>
<td>Electrode glass</td>
<td>World Precision Instruments</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Filters</td>
<td>Filter</td>
<td>Whatman</td>
<td>6789-0402</td>
<td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td>
</tr>
<tr>
<td>IR camera</td>
<td>Micropscope mounted camera</td>
<td>Sony</td>
<td>SPT-M324</td>
<td>Extrawave HAD, B7W video camera</td>
</tr>
<tr>
<td>Picrotoxin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1675</td>
<td>&nbsp;</td>
<tr>
<td>Strychnine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0532</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole-4-acetic acid sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I7013</td>
<td>&nbsp;</td>
<tr>
<td>Larval tiger salamanders</td>
<td>Animal</td>
<td>Charles E. Sullivan (Nashville, TN)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

نستخدم المشبك التصحيح خلية كاملة لدراسة تقنية الدارات متشابك التي ترتكز عليها معالجة المعلومات البصرية في الشبكية. في هذا الفيديو ، فإننا سوف دليل لكم من خلال عملية تنفيذ كامل الخلية تسجيلات ضوء التيارات أثار الخلايا الفردية في إعداد شريحة الشبكية. نستخدم نمر المائية السمندل كنموذج للحيوانات. نبدأ بوصف تشريح العين وكيفية عرض الشرائح هي التي شنت للتسجيلات الكهربية. مرة واحدة يتم وضع شريحة في غرفة تسجيل ، ونحن لشرح كيفية تنفيذ كامل الخلية التسجيلات المشبك الجهد. نحن المشروع ثم محفزات بصرية وعلى المستقبلات الضوئية في لانتزاع شريحة ضوئية أثار ردود الحالي. خلال تسجيل يروي لنا شريحة مع وكلاء الدوائية ، حيث نظام الارواء 8 قنوات تسمح لنا التبديل بسرعة بين وكلاء مختلفة. ويستخدم على نطاق واسع في إعداد شريحة الشبكية للتسجيلات المشبك التصحيح في شبكية العين ، لا سيما لدراسة الخلايا أو عديم الاستطالات القطبين ، والتي لا يمكن الوصول إليها في إعداد كامل جبل.

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

هذا الفيديو يبين عملية خلية كاملة التسجيلات المشبك الجهد في شريحة الشبكية من السمندل النمر المائية. نبدي استعدادا لشريحة وكذلك كيفية تنفيذ تسجيلات المشبك التصحيح خلال التحفيز البصري لشبكية العين.

مستدعى خلية كاملة تسجيلات الضوء التيارات متشابك مثير في شريحة الشبكية

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Research

JoVE Journal

Biology

12.7K Views.  Boston University. هذا الفيديو يبين عملية خلية كاملة التسجيلات المشبك الجهد في شريحة الشبكية من السمندل النمر المائية. نبدي استعدادا لشريحة وكذلك كيفية تنفيذ تسجيلات المشبك التصحيح خلال التحفيز البصري لشبكية العين.

Video: Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

إعداد شريحة الأفقي للشبكية

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

تسجيلات كاملة من خلية دماغ ذبابة الفاكهة الكبار

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

مكونات رئيسية مجهر الضوء

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

تسجيلات لتنشيط الدائرة العصبية في التصرف بحرية حيوانات

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

تسجيلات شفط وحيدة الخلية المستقبلة من المخروط ماوس

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

خلية كاملة تسجيل النسخة الكالسيوم المنشط الكالسيوم (CRAC) التيارات في الخلايا اللمفية تي الإنسان

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

دراسة التحلل البروتيني B من السيكلين على مستوى خلية واحدة في الجامعة السكان الخلية

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

أسلوب الثقافة شريحة لوبعد تطور الجراثيم الأسنان في يزدرع الثقافة

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

طريقة لتقييم الآثار المترتبة على مجموعة من موجات وشدة الأحمر / الأشعة تحت الحمراء القريبة العلاج بالضوء على الإجهاد التأكسدي<em&gt; في المختبر</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...