وأيد هذا العمل من قبل المعاهد الوطنية للصحة HL109478 (KNF). يعترف الكتاب وشكرا جينا كيم وجوان تايلور لمساعدتها في عزل والمحافظة على PASMC في الثقافة.

<ol>
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Physiol. 283, L1103-L1109 (2002).</li><li>Aslam, M., 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=Bone+marrow+stromal+cells+attenuate+lung+injury+in+a+murine+model+of+neonatal+chronic+lung+disease.">Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease.</a> Am. J. Respir. Crit. Care Med. 180, 1122-1130 (2009).</li><li>Balasubramaniam, V., 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=Bone+marrow-derived+angiogenic+cells+restore+lung+alveolar+and+vascular+structure+after+neonatal+hyperoxia+in+infant+mice.">Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 298, L315-L323 (2010).</li><li>de Visser, Y. 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=Phosphodiesterase-4+inhibition+attenuates+pulmonary+inflammation+in+neonatal+lung+injury.">Phosphodiesterase-4 inhibition attenuates pulmonary inflammation in neonatal lung injury.</a> Eur. Respir. J. 31, 633-644 (2008).</li><li>de Visser, Y. 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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=Brief+hyperoxia+increases+mitochondrial+oxidation+and+increases+phosphodiesterase+5+activity+in+fetal+pulmonary+artery+smooth+muscle+cells.">Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.</a> Antioxid. Redox Signal. 17, 460-470 (2012).</li><li>Waypa, G. B., 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=Hypoxia+triggers+subcellular+compartmental+redox+signaling+in+vascular+smooth+muscle+cells.">Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.</a> Circ. Res. 106, 526-535 (2010).</li><li>Waypa, G. B., 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=Superoxide+Generated+at+Mitochondrial+Complex+III+Triggers+Acute+Responses+to+Hypoxia+in+the+Pulmonary+Circulation.">Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation.</a> Am. J. Respir. Crit. Care Med. 187, 424-432 (2013).</li><li>Marshall, C., Mamary, A. J., Verhoeven, A. J., Marshall, 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=Pulmonary+artery+NADPH-oxidase+is+activated+in+hypoxic+pulmonary+vasoconstriction.">Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.</a> Am. J. Respir. Cell Mol. Biol. 15, 633-644 (1996).</li><li>Farrow, K. 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=Superoxide+Dismutase+and+Inhaled+Nitric+Oxide+Normalize+Phosphodiesterase+5+Expression+and+Activity+in+Neonatal+Lambs+with+Persistent+Pulmonary+Hypertension.">Superoxide Dismutase and Inhaled Nitric Oxide Normalize Phosphodiesterase 5 Expression and Activity in Neonatal Lambs with Persistent Pulmonary Hypertension.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 299, L109-L116 (2010).</li></ol>

يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

في هذا المخطوط، وصفنا للمرة الأولى عزلة PASMC من الفئران في P7، P14، P21 و. من أجل تحقيق هذا الهدف، وغرست في الطين من الاغاروز و 0.2 ميكرومتر جزيئات الحديد من خلال RV في السلطة الفلسطينية. ونظرا لصغر حجم جزيئات الحديد، فإنها لا يمكن أن تمر عبر السرير الشعري الرئوي، وبالتالي فهي...

ارتفاع ضغط الدم الرئوي أمر طبيعي خلال الحياة داخل الرحم منذ المشيمة بمثابة الجهاز الرئيسي لتبادل الغازات ويتم توزيعها فقط 10٪ من النتاج القلبي من خلال السرير الوعائي الرئوي. في الرحم والضغوط الرئوي تشبه الضغوط النظامية نظرا لارتفاع المقاومة الوعائية الرئوية . كما تقدم الحمل، وهناك نمو سريع للسلطة الفلسطينية الصغيرة داخل الرئة، وإعداد الجنين لزيادة كبيرة في تدفق الدم الرئوي الذي يحدث عند الولادة 1. عند فشل عملية الانتقال في الفترة المحيطة بالولادة الطبيعية لدى الرضع على المدى القريب والمدى الكامل، والنتيجة هي ارتفاع ضغط الدم الرئوي الثابتة من الأطفال حديثي الولادة (PPHN). PPHN هو متلازمة سريرية بسبب العديد من الأمراض الكامنة مختلفة. ومع ذلك، كل من هؤلاء الرضع بسمات الفسيولوجية المرضية الشائعة مثل ارتفاع المقاومة الوعائية الرئوية، نقص الأكسجة، وتحويلة من اليمين إلى اليسار من تدفق الدم عبر اتصالات الجنين المستمرة مثل القناة الشريانية أو للآمين البيضوية. PPHN يؤثر 2-6 لكل 1،000 ولادة حية وينقل مخاطر 8-10٪ من وفيات فضلا كبيرا على المدى القصير وعلى المدى الطويل 2 الاعتلال. بالإضافة إلى ذلك، قد منخفضة جدا الوزن عند الولادة الخدج تطوير ارتفاع ضغط الدم الرئوي نتيجة لمرضهم الرئة الكامنة. وأمراض الرئة الكامنة الأكثر شيوعا من الأطفال الخدج هو خلل التنسج القصبي الرئوي. في حين أن المخاطر الكلية للبرميل في اليوم يرتبط مع العمر الحملي ووزن الولادة، يبقى من غير الواضح لماذا مجموعة فرعية من هؤلاء الرضع تطور ارتفاع ضغط الدم الرئوي هامة وكيفية التعامل مع هؤلاء الأطفال بشكل مناسب. النتائج السيئة، بما في ذلك الإقامة في المستشفى لفترات طويلة وزيادة معدل الوفيات، شائعة 3-6. تاريخيا، استخدمت الأغنام PASMC الجنين أو الخنازير PASMC الجنين من الحيوانات السليمة لدراسة مسارات الإشارات المشاركة في عملية الانتقال الأوعية الدموية الرئوية الطبيعية بعد الولادة. عادة يتم عزل هذه من الجيل الخامس المقاومة الفلسطينية منن الأغنام أو الخنازير الجنين التي يتم تسليمها والموت الرحيم قبل أي التنفس العفوي 7-9. بالإضافة إلى ذلك، بعض المحققين عزلت والاستفادة منها PASMC من كبار السن قليلا ويتنفس بشكل عفوي الحملان والخنازير في 3 أيام، 2 أسابيع، و4 أسابيع 10-12. وفي الآونة الأخيرة، بعض المجموعات عزلت بنجاح والاستفادة منها PASMC معزولة عن الحملان مع PPHN لدراسة خللا في مسارات إشارات في حالة المرض 13-17. وقد أثبتت هذه الخلايا لتكون أداة قيمة لدراسة مسارات الإشارات التي تشكل عاملا حاسما في كل من الأوعية الدموية الرئوية طبيعية والمريضة على المدى القريب والبعيد. ومع ذلك، فإنها لا تعطي نظرة ثاقبة في مسارات الإشارات أثرت في الأطفال الخدج المصابين بارتفاع ضغط الدم الرئوي. كما أنها لا تسمح إمكانيات التلاعب الجيني ينظر في نماذج الماوس من المرض. منذ فترة طويلة تستخدم الجرذان والفئران نماذج لنموذج برميل يوميا ويجري استخدامها في الآونة الأخيرة إلى نموذج HY الرئويpertension الناتجة عن برميل يوميا 18-22. الفئران حديثي الولادة هي تحريضية للعمل مع نظرا لحجم أكبر، لكنها تعاني أيضا من عدم وجود إمكانية التعديل الوراثي. تم الحيوانات المعدلة وراثيا المستخدمة على نطاق واسع للتحقيق في الآثار المترتبة على أهداف جين معين على فسيولوجيا الحيوان كله في الفئران حديثي الولادة، ولكن حتى الآن لا أحد سبق له معزولة بنجاح PASMC من هذه الفئران الصغيرة. من خلال عزل PASMC، ويمكن الحصول على مزيد من المعلومات حول كيفية تغيير مسارات في الاستجابة للمؤثرات البيئية و / أو التعديل الوراثي وتحديدا في العضلات الشريان الرئوي على نحو سلس. بالإضافة إلى ذلك، يمكن تصوير الحية PASMC في الوقت الحقيقي لدراسة التغيرات السريعة في الجزيئات يشير المفتاح مثل الكالسيوم وأنواع الاكسجين التفاعلية 23-25. وصفنا في الآونة الأخيرة عزلة الناجح لPASMC من فئران بالغة باستخدام تباين أسلوب مارشال وآخرون. 26 تستخدم لعزل الفئران PASMC 23،25،26. نحن الآن تكيفت علىتمديد الثانية هذه التقنية على الفئران الصغيرة 7-21 أيام من العمر (P7، P14، P21 و). والقيد الرئيسي لهذه التقنية الجديدة العزلة PASMC هو أنه يتطلب الفئران متعددة لتوليد خلايا كافية لإجراء التجارب وأن الخلايا تنمو ببطء شديد، والذي هو سمة من خلايا العضلات الملساء الأولية. وعلى الرغم من هذه القيود، ونحن نعتقد أن هذا الأسلوب لعزل PASMC الماوس حديثي الولادة تسمح للتحقيق تعزيز مسارات الإشارات الرئيسيين المشاركين في تطوير ارتفاع ضغط الدم الرئوي، ويمثل تقدما كبيرا في هذا المجال.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Bard Parker surgical blade handle</td>
			<td>BD</td>
			<td>371030</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel surgical blades #10 (sterile)</td>
			<td>Miltex</td>
			<td>4-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (3 ml and 5 ml, sterile)</td>
			<td>BD</td>
			<td>309657 and 306646</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 G, sterile)</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocatheter (24 G, sterile)</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoject blunt cannula (15 G)</td>
			<td>Kendall</td>
			<td>SWD202314Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Fisher Scientific</td>
			<td>NC9782896</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynal magnet particle collector</td>
			<td>Invitrogen</td>
			<td>120-01D</td>
			<td>This is a critical tool for the protocol.</td>
		</tr>
		<tr>
			<td>Tissue culture plates (35 mm, 60 mm, and 10 cm, sterile)</td>
			<td>BD</td>
			<td>353001, 353004, and 353003</td>
			<td>Any brand of tissue culture plates will be fine.</td>
		</tr>
		<tr>
			<td>Iris Scissors (4 &#189; inch stainless steel)</td>
			<td>American Diagnostic Corporation</td>
			<td>3424</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (4 inch stainless steel)</td>
			<td>Quick Medical</td>
			<td>L5-5004</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>M199 media</td>
			<td>Mediatech</td>
			<td>10-060-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>TX16777-164NWU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone/Thermo Scientific</td>
			<td>SH3091003</td>
			<td>Heat inactivate at 55 &#176;C for 45 min. For consistency in results, lot match all serum and obtain from same vendor.</td>
		</tr>
		<tr>
			<td>Iron particles (iron (II, III) oxide powder)</td>
			<td>Aldrich Chemical Company</td>
			<td>#31,006-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (made to 80 U/ml)</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butlet Schein</td>
			<td>NDC 11695-6776-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE2000-U with a Cascade Photometrics 12-bit camera</td>
			<td>Nikon</td>
			<td>TE2000-U</td>
			<td>Any good light microscope will be fine to observe PASMC in culture.</td>
		</tr>
		<tr>
			<td>Anti-desmin antibody</td>
			<td>Sigma</td>
			<td>D-8281</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Anti-smooth muscle myosin</td>
			<td>Biomedical Technologies</td>
			<td>BT-562</td>
			<td>Use at 1:2,000 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Rhodamine-red anti-rabbit secondary</td>
			<td>Molecular Probes/Invitrogen</td>
			<td>R-6394</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE-300 fluorescent microscope and Cool Snap digital camera</td>
			<td>Nikon</td>
			<td>TE300</td>
			<td>Any good epifluorescence microscope will be fine for immunostaining.</td>
		</tr>
		<tr>
			<td>Cyclic nucleotide phosphodiesterase assay kit</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK800-0001</td>
			<td>This is the only colorimetric PDE enzyme activity assay available.</td>
		</tr>
		<tr>
			<td>Sildenafil</td>
			<td>Sigma</td>
			<td>PZ-0003</td>
			<td>A PDE5-selective inhibitor is required for the PDE enzyme activity to determine specificity of cGMP hydrolysis.</td>
		</tr>
	</tbody>
</table>

isolation of pulmonary artery smooth muscle cells from neonatal mice

ارتفاع ضغط الدم الرئوي هو سبب كبير من المراضة والوفيات عند الأطفال الرضع. تاريخيا، كانت هناك دراسة كبيرة من مسارات الإشارات المشاركة في الأوعية الدموية تقلص العضلات الملساء في PASMC من الأغنام الجنين. بينما الأغنام جعل نموذجا ممتازا من ارتفاع ضغط الدم الرئوي المدى، أنها مكلفة للغاية وتفتقر إلى الاستفادة من التلاعب الوراثي الموجود في الفئران. على العكس، كان عدم القدرة على عزل PASMC من الفئران وجود قيود كبيرة من هذا النظام. نحن هنا وصف عزلة الثقافات الأولية من الماوس PASMC من P7، P14، P21 والفئران باستخدام الاختلاف من هذه التقنية هو موضح سابقا من مارشال وآخرون. ال 26 التي كانت تستخدم سابقا لعزل PASMC الفئران. هذه PASMC الفئران تمثل أداة جديدة لدراسة مسارات إشارات في فترة الوليد. لفترة وجيزة، والطين من 0.5٪ (W / V) + الاغاروز هي التي غرست جزيئات الحديد 0.5٪ في M199 سائل الإعلام في السرير الوعائي الرئوي عبر البطين الأيمن (RV). الجزيئات الحديد هي 0.2 ميكرومتر في القطر وليس يمكن أن تمر عبر السرير الشعري الرئوي. وهكذا، فإن النزل الحديد في الشرايين الرئوية الصغيرة (PA). وتضخم الرئتين مع الاغاروز، إزالتها وفصلها. يتم سحبها في الأوعية المحتوية على الحديد إلى أسفل مع المغناطيس. بعد كولاجيناز (80 U / مل) العلاج ومزيد من التفكك، يتم وضع السفن في طبق زراعة الأنسجة في M199 سائل الإعلام التي تحتوي على 20٪ مصل بقري جنيني (FBS)، والمضادات الحيوية (M199 وسائل الإعلام كاملة) للسماح الهجرة الخلية على طبق ثقافة . هذه لوحة من الخلايا الأولية هي مزيج من الخلايا الليفية 50-50 وPASMC. وهكذا، فإن هدم الإجراء يتكرر عدة مرات لتحقيق السكان PASMC أكثر نقاء وإزالة أي الحديد المتبقية. يتم تأكيد هوية الخلايا العضلية الملساء التي كتبها المناعية لسلسة العضلات الميوسين وdesmin.

أثناء وبعد العزلة، ويتم فحص كل PASMC بواسطة المجهر الضوئي والمناعية لسلسة علامات الخلية العضلية. بواسطة المجهر الضوئي في البروتوكول في وقت مبكر، وينظر PASMC المهاجرة على طبق زراعة الأنسجة من الحديد الصغيرة التي تحتوي على الأوعية (الشكل 1A). بعد تجميع لوحات واحد م?...

Watch this Scientific Journal Video about Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice at JoVE.com

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

وقد وضعنا رواية وتقنية استنساخه لعزل الثقافات الأولية من الشريان الرئوي خلايا العضلات الملساء (PASMC) من الفئران في سن P7، مما يتيح دراسة أفضل للمسارات الإشارات المشاركة في حديثي الولادة العضلات الملساء انكماش الخلايا والاسترخاء.

عزل الشريان الرئوي خلايا العضلات الملساء من الفئران حديثي الولادة

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

isolation-of-pulmonary-artery-smooth-muscle-cells-from-neonatal-mice

Research

JoVE Journal

Biology

18.1K Views.  Northwestern University Feinberg School of Medicine. وقد وضعنا رواية وتقنية استنساخه لعزل الثقافات الأولية من الشريان الرئوي خلايا العضلات الملساء (PASMC) من الفئران في سن P7، مما يتيح دراسة أفضل للمسارات الإشارات المشاركة في حديثي الولادة العضلات الملساء انكماش الخلايا والاسترخاء.

Video: Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

تنسيق كا 2 + الكشف العابر في العضلات الملساء

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

عزل الشريان السري الإنسان خلايا العضلات الملساء (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

العزلة والثقافة في الخلايا البطانية الرئوي من الفئران حديثي الولادة

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

الميتوكوندريا من عزل العضلات والهيكل العظمي

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

العزلة وثقافة فردية وMyofibers زنزاناتهم القمر الصناعي من العضلات الهيكلية الكبار

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

العزلة والثقافة، وزرع خلايا العضلات الأقمار الصناعية

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

عزل خلايا الفئران صمام غشائي

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

عزل الفئران التاجي الأوعية الدموية خلايا العضلات الملساء

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

قياس انتشار خلايا العضلات الملساء الوعائية باستخدام الكيمياء فوق

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

عزل القلب البطيني البشري من شرائح عضلة القلب المقطّع بالهزاز

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...