The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

أجريت الدراسات على الحيوانات في إطار البروتوكول التي وافق عليها رعاية الحيوان المؤسسية واستخدام اللجنة في معهد البوليتكنيك فرجينيا وجامعة ولاية. 1. إعداد (الوقت: ~ 45 دقيقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مخازن تجميد ذوبان الجليد من 0.25٪ التربسين، عزل عازلة للالميتوكوندريا (IBM) 1 وIBM2 في حمام مائي 37 ° C. </li><li style=";text-align:right;direction:rtl"> شطف الأواني الزجاجية وأدوات تشريح في الايثانول 70٪ تليها مياه عالية النقاء. </li><li style=";text-align:right;direction:rtl"> إعداد 0.05٪ محلول التربسين من 0.25٪ التربسين الأسهم عن طريق تمييع 1 جزء التربسين في 4 أجزاء IBM1. </li><li style=";text-align:right;direction:rtl"> مزيج البروتيني والفوسفاتيز المانع كوكتيل مع العازلة تحلل الخلية (1: 100 نسبة) في أنبوب microcentrifuge 1.5 مل مع غطاء المسمار. </li><li style=";text-align:right;direction:rtl"> قسامة 5 مل (5 مل / الماوس) من 0.05٪ التربسين في أنبوب من البلاستيك 15 مل. </li><li style=";text-align:right;direction:rtl"> تعيين بالمواتير الخالط الأنسجة إلى 80 دورة في الدقيقة. </li></ol> 2. عزل العضلات والهيكل العظمي الميتوكوندريا (الوقت: ~ 90 دقيقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء الماوس عن طريق CO 2 </suب&gt; استنشاق، تليها خلع عنق الرحم. </li><li style=";text-align:right;direction:rtl"> إزالة العضلات الحمراء من عضلات الفخذ والساق العضلات، والذي يتضمن النعلية (75-100 ملغ ~ الكل) كما هو موضح في الخطوات التالية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قشر الجلد نحو الماوس. </li><li style=";text-align:right;direction:rtl"> إزالة لوحة الدهون أكثر من نقطة الأصل الرباعية الرؤوس. قطع العضلة رباعية الرؤوس التي يتم تركيبها على الرضفة مع مقص يميل غرامة. ملاحظة: يتم التعرف على عضلات الفخذ التي كتبها الوضعية التشريحية على الجزء الأمامي من الفخذ القريبة. </li><li style=";text-align:right;direction:rtl"> قص ببطء صفاق بين العظام وعضلات الفخذ، مع تجنب الشريان الفخذي، لتحرير عضلات الفخذ من العظم. قطع الوتر مع مقص يميل غرامة عند نقطة الأصل على عظم الفخذ لتحرير عضلة الفخذ ووضع عضلة الفخذ في برنامج تلفزيوني برود. </li><li style=";text-align:right;direction:rtl"> إزالة الأنسجة الدهنية واضحة على عضلات الفخذ مع مقص. الوجه عضلات الفخذ بحيث تكون جزء من العضلات التي كانت تغمر عظم الفخذ يواجه شص. فتح عضلة الفخذ مع ملقط في حركة تأجيج. إزالة اثنين من مرئية أجزاء العضلات الحمراء من كل شحمة الأذن مع مقص يميل الجميلة ومكان في دورق يحتوي على 5 مل من برود IBM1. ملاحظة: العضلة الرباعية الرؤوس تظهر كما فصين مع شريط من العضلات الحمراء بالقرب من الحافة الجانبية من كل شحمة الأذن. </li><li style=";text-align:right;direction:rtl"> قطع الجلد المغطي في وتر العرقوب مع غرامة يميل مقص وقشر الجلد نحو الماوس. قطع وتر العرقوب المكشوفة وقشر العضلات نحو جسم الفأر. </li><li style=";text-align:right;direction:rtl"> قطع وتر في اللقم الجانبية وسطي من عظم الفخذ مع مقص يميل غرامة لتحرير الساق ووضع الساق تعلق على الأوتار في PBS المبردة. </li><li style=";text-align:right;direction:rtl"> الوجه الساق مرارا وتقشر عضلة النعلية ووضع في دورق يحتوي على 5 مل من برود IBM1. مروحة فتح الساق. إزالة الأجزاء الثلاثة البصرية الحمراء العضلات (قطعتين الحمراء الجانبية وسطي واحد السطحية) مع غرامة يميل مقصالصورة ونقلها إلى دورق يحتوي على 5 مل من برود IBM1. </li></ol></li><li style=";text-align:right;direction:rtl"> إزالة ~ 75-100 ملغ أخرى من العضلات الحمراء من الساق الأخرى من الفأرة (كما هو موضح في الخطوة 2.2) ووضع في أنبوب microcentrifuge 1.5 مل مع البروتيني والفوسفاتيز المانع كوكتيل وعازلة تحلل الخلية (50 ملي تريس، HCL، 1 ملي EDTA، 150 ملي كلوريد الصوديوم، 1٪ كبريتات دوديسيل الصوديوم، 0.5٪ deoxycholate الصوديوم، 1٪ البولي أوكسي إتيلين (9) nonylphenylether، تشعبت وعلى الفور فلاش-تجميد هذه العينة الثانية في النيتروجين السائل لاستخدامها لاحقا في غرب النشاف (راجع الخطوة 6.1). </li><li style=";text-align:right;direction:rtl"> وضع سطح البلاستيك شقة قبل المبردة على الجليد في دلو كبير. صب قطرة من IBM1 على سطح البلاستيك واستخدام الملقط لوضع كل من العضلات الحمراء مقطوع (الخطوة 2.2.4 و2.2.7) في IBM1 الحبرية. تخطر على الأنسجة العضلية لمدة 2 دقيقة واحدة باستخدام 3 شفرات الحلاقة حافة بواسطة شفرات الحلاقة تتغير كل 40 ثانية. </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة المفروم إلى دورق جديد مع 5 مل من IBM1 جديدة عن طريق الضغط على سطح البلاستيك أكثر من عشرةالبريد كوب وتجريف العضلات في الكأس بشفرة حلاقة. خذ هذا الحل واستنزاف من خلال مصفاة الخلية 100 ميكرون وضعت على 50 مل أنبوب مخروطي الشكل. </li><li style=";text-align:right;direction:rtl"> وصمة عار الأنسجة مع ممسحة مهمة حساسة ومن ثم نقلها إلى 5 مل من محلول التربسين 0.05٪. استخدام ملاقط لإزالة الأنسجة من المساحات المهمة. </li><li style=";text-align:right;direction:rtl"> احتضان الأنسجة العضلية على الجليد لمدة 30 دقيقة في 0.05٪ محلول التربسين. </li><li style=";text-align:right;direction:rtl"> تدور أنبوب مخروطي 15 مل تحتوي على خليط التربسين / العضلات في 200 x ج لمدة 3 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> صب طاف التربسين في حاوية النفايات و resuspend بيليه مع 3 مل من الجليد IBM1 البرد. نقل الأنسجة إلى 45 مل أنبوب زجاجي الخالط. شطف أنبوب مخروطي 15 مل مع 1.5 مل IBM1 آخر لجمع أي عينة المتبقية وهذا إضافة إلى أنبوب زجاجي الخالط. </li><li style=";text-align:right;direction:rtl"> وضع أنبوب زجاجي الخالط الى شغل في منتصف الطريق كوب أو حاوية بلاستيكية مع الثلج حتى ينتقل الخالط الزجاج الحد الأدنى داخل الكأس. </li><li&gt; تجانس الخليط الأنسجة / IMB1 مع مدقة PTFE تعلق على بالمواتير الأنسجة الخالط مع 10 تمريرات في 80 دورة في الدقيقة. عقد أسفل كل تمريرة ل~ 2 ثانية. </li><li style=";text-align:right;direction:rtl"> نقل جناسة الأنسجة لأنبوب 15 مل مخروطي الشكل الجديد وشطف أنبوب زجاجي الخالط مع 6.5 مل من IBM1. صب IBM1 إلى 15 مل أنبوب مخروطي يحتوي على جناسة الأنسجة. </li><li style=";text-align:right;direction:rtl"> تدور أنبوب مخروطي 15 مل في 700 غ لمدة 10 دقيقة على 4 درجات مئوية. صب بلطف طاف في كوب قوة عالية أنبوب الطرد المركزي وتجاهل بيليه. </li><li style=";text-align:right;direction:rtl"> تدور طاف من الخطوة السابقة في 8000 x ج لمدة 10 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة طاف IBM1 وإعادة تعليق بيليه بإضافة ببطء 500 ميكرولتر من IBM2. قطع نهاية طرف ماصة والتجانس بلطف بيليه في IBM2 مع الخلط والتقليب الاقتراحات. إضافة 4.5 مل أخرى من IBM2 إلى الخليط بعد تعليق بيليه بالكامل. ملاحظة: تجنب الإفراط في سحن حين كسر قطعة بيليه أكبر لرله قد يؤدي إلى تلف أغشية الميتوكوندريا. </li><li style=";text-align:right;direction:rtl"> تدور IBM2 / الأنسجة جناسة في 8000 x ج لمدة 10 دقيقة على 4 درجات مئوية. ملاحظة: يمكن تكرار هذه الخطوة في نظيفة قوة عالية أنبوب الطرد المركزي لأدق تقدير البروتين الميتوكوندريا معزولة في الخطوة 4 منذ المتبقي BSA قد تسهم في محتوى البروتين الكلي. </li><li style=";text-align:right;direction:rtl"> إزالة طاف وبلطف، ولكن تماما اعادة تعليق بيليه بإضافة اثنين 25 الزيادات ميكرولتر من IBM2 مع طرف ماصة مع وجهة نظرها قطع. يحرك بلطف وتخلط بيليه بعد كل إضافة 25 ميكرولتر من IBM2. ضع هذا المخزون الميتوكوندريا على الجليد. </li></ol> 3. تجانس النسيج كله المحللة (الوقت: ~ 45 دقيقة، ويمكن أن يتم ذلك خلال الخطوة 7) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة العضلات من أن وضعت في النيتروجين السائل من الخطوة 2.3 واحتضان في درجة حرارة الغرفة حتى إذابة تماما. </li><li style=";text-align:right;direction:rtl"> تخطر على الأنسجة ل~ 30 ثانية على الجليد مع مقص يميل غرامة. </li><li style=";text-align:right;direction:rtl"> إضافة إلى اثنين من المجارف 100 ميكرولتر من Zircoأكسيد nium الخرز لأنبوب microcentrifuge 1.5 مل مع غطاء المسمار من الخطوة 3.2 الذي يحتوي على الأنسجة المفروم. تجانس الأنسجة في الخالط الأنسجة طاحونة حبة باستخدام 2-3 5 دورات دقيقة في إعداد سرعة 4-6 (الإعداد المتوسط). </li><li style=";text-align:right;direction:rtl"> تدور خليط من الخطوة 3.3 في 12000 x ج لمدة 10 دقيقة على 4 درجات مئوية. إزالة طاف باستخدام 1000 ميكرولتر ماصة بعد زيادة ونقصان ونقل في أنبوب microcentrifuge 1.5 مل. تجاهل بيليه ووضع طاف على الجليد. </li></ol> 4. البروتين تحديد (الوقت: ~ 30 دقيقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد تركيز البروتين في الأنسجة المحللة كله (الخطوة 3.4) والأسهم الميتوكوندريا (الخطوة 2.17) باستخدام BCA عدة فحص البروتين وفقا لمواصفات الشركة المصنعة. </li></ol> 5. الميتوكوندريا غشاء النزاهة. سينسيز سيترات (CS) آخر (الوقت: ~ 15 دقيقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل اثنين منفصلة 1: 20 التخفيفات من الأوراق المالية الميتوكوندريا في المياه عالية النقاء. إضافة0.1٪ من تريتون 100-X لعينة واحدة ويصوتن ذلك على إعداد منخفض (765 واط، سعة 2). ترك التخفيف الآخرين على الجليد. العودة العينة إلى الجليد بعد صوتنة. </li><li style=";text-align:right;direction:rtl"> إجراء فحص سينسيز سترات مع كل من غير sonicated وsonicated التخفيفات الميتوكوندريا باستخدام الفحص الطيفي كما هو موضح سابقا 11،12. </li><li style=";text-align:right;direction:rtl"> تحديد النسبة المئوية للأغشية الميتوكوندريا سليمة باستخدام المعادلات التالية: (غير sonicated النشاط CS ÷ Sonicated النشاط CS) * 100 </li></ol> 100- N (في المئة حققت من فوق) 6. الميتوكوندريا الجودة العزلة. الغربية التنشيف (الوقت: وفقا لبروتوكول مختبر) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أداء النشاف الغربي على المحللة النسيج كله والميتوكوندريا معزولة كما هو موضح سابقا (12). ملاحظة: استخدام الأجسام المضادة الأولية لGAPDH (1: 1000 تخفيف في 5٪ BSA / TBS-T) وCOXIV (1: 1000 تخفيف في 5٪ الخالي من الدسمالحليب / TBS-T)، يليه مكافحة الماعز، والأرانب الأجسام المضادة الثانوية، على التوالي (1: 10000). </li></ol> 7. Respirometric الفحص تشغيل (الوقت: ~ 90 دقيقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أداء المقايسات respirometric المطلوب باستخدام صفيحة ميكروسكوبية أساس O 2 تكنولوجيا قياس الاستهلاك كما هو موضح سابقا (انظر المادة مصاحب وروجرز وآخرون 8). </li><li style=";text-align:right;direction:rtl"> تحديد نسبة السيطرة التنفسية (RCR) بتقسيم الدولة 3 في الدولة 4O (RCR) أو تقسيم 3U الدولة من قبل دولة 4O. استخدام أي منتصف (متوسط) نقطة أو أعلى (الدولة 3) وأدنى معدلات (4O الدولة) من آثار نقطة إلى نقطة عند تحديد RCR. </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> JoVE. , (2011).</li><li>Gross, V. S., 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=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Anal. Biochem. 418, 213-223 (2011).</li><li>Krieger, D. A., Tate, C. A., McMillin-Wood, J., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+rat+skeletal+muscle+mitochondria+after+exercise+and+immobilization.">Populations of rat skeletal muscle mitochondria after exercise and immobilization.</a> J. Appl. Physiol.: Respir., Envir. and Ex. Physiol. 48, 23-28 (1980).</li><li>Asmann, Y. W., 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=Skeletal+muscle+mitochondrial+functions,+mitochondrial+DNA+copy+numbers,+and+gene+transcript+profiles+in+type+2+diabetic+and+nondiabetic+subjects+at+equal+levels+of+low+or+high+insulin+and+euglycemia.">Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.</a> Diabetes. 55, 3309-3319 (2006).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods Enzymol. 457, 349-372 (2009).</li></ol>

George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

الطرق الواردة في هذه الوثيقة تقدم وصفا مفصلا لإجراء العزلة الميتوكوندريا من كميات ضئيلة (~ 75-100 ملغ) من الماوس العضلات والهيكل العظمي. هذا الإجراء العزلة هي قادرة على تسفر عن أداء عالية، الميتوكوندريا النقي (~ 450 ميكروغرام) كما يتضح من O 2 معدلات الاستهلاك، والقيم R...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</td>
		</tr>
		<tr>
			<td>Tris/HCl</td>
			<td>Promega</td>
			<td>H5123</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Promega</td>
			<td>H5135</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma Aldrich</td>
			<td>63559</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200-056</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Chloride 
			White Crystals or Crystalline Powder 
			&#8805;99.0 %</td>
			<td>Fisher Scientific</td>
			<td>BP3581</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L3771&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Polyoxyethylene (12) nonylphenyl ether, branched</td>
			<td>Sigma Aldrich</td>
			<td>238651</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Single Edge Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Falcon- 100 uM Nylon Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>352360</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>GAPDH antibody (1D4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-59540</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Anti- COXIV antibody</td>
			<td>Cell Signaling</td>
			<td>4844s</td>
			<td>Any mitochondrial inner membrane protein will suffice</td>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pyruvic Acid, 98%</td>
			<td>Sigma Aldrich</td>
			<td>107360</td>
			<td>Store at 4&#176;C,pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>Succinic Acid</td>
			<td>Sigma Aldrich</td>
			<td>S9512</td>
			<td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>L(-) Malic Acid, BioXtra, &#8805;95%</td>
			<td>Sigma Aldrich</td>
			<td>M6413</td>
			<td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma Aldrich</td>
			<td>R8875</td>
			<td>Store at room temperature</td>
		</tr>
	</tbody>
</table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

مختلة الميتوكوندريا العضلات والهيكل العظمي تلعب دورا في عملية التمثيل الغذائي تغير لاحظ مع الشيخوخة والبدانة ومرض السكري من النوع الثاني. المقايسات respirometric الميتوكوندريا من التحضيرات الميتوكوندريا المعزولة تسمح لتقييم وظيفة الميتوكوندريا، وكذلك تحديد آلية (ق) للعمل الأدوية والبروتينات التي تعدل الأيض. وغالبا ما تتطلب إجراءات العزلة الحالية كميات كبيرة من الأنسجة لانتاج الميتوكوندريا عالية الجودة اللازمة لفحوصات respirometric. الطرق الواردة في هذه الوثيقة تصف كيف عالية الجودة الميتوكوندريا المنقى (~ 450 ميكروغرام) يمكن عزله عن كميات ضئيلة (~ 75-100 ملغ) من الماوس العضلات والهيكل العظمي للاستخدام في قياسات الجهاز التنفسي إنتاجية عالية. توصلنا إلى أن طريقة عزلتنا ينتج 92.5 ± 2.0٪ الميتوكوندريا سليمة من خلال قياس النشاط سينسيز سترات طيفيا. بالإضافة إلى ذلك، أدى تحليل لطخة غربية في الميتوكوندريا المعزولة في التعبير خافت من cytosoالبروتين يسانس، GAPDH، والتعبير القوي للبروتين الميتوكوندريا، COXIV. عدم وجود الفرقة GAPDH بارزة في الميتوكوندريا المعزولة يدل على التلوث القليل من مصادر غير الميتوكوندريا أثناء إجراء العزل. الأهم من ذلك، وقياس معدل استهلاك O 2 مع لوحة الصغيرة القائمة على التكنولوجيا وتحديد نسبة السيطرة التنفسية (RCR) لفحوصات respirometric المترابطة معارض البالغ الاقتران (RCR؛ &lt;6 لجميع المقايسات) والميتوكوندريا وظيفية. في الختام، إضافة خطوة تنميق منفصلة وسمحت تخفيض كبير بالمواتير سرعة تجانس طريقة عنها سابقا عزلة جودة عالية والميتوكوندريا تنقيته من كميات صغيرة من الماوس العضلات والهيكل العظمي الذي ينتج في الميتوكوندريا بالإضافة جدا أن نتيجة التنفس مع وظيفة عالية خلال صفيحة ميكروسكوبية مقرها المقايسات respirometirc.

يخدم النشاط سينسيز سترات كمقياس للسلامة الغشاء منذ سينسيز سترات يقع في غشاء الميتوكوندريا الداخلية، وبالتالي يجب أن لا تكون موجودة في تعليق الميتوكوندريا مع الأغشية سليمة الشكل 1 يمثل النشاط سينسيز سيترات في عينات غير sonicated الميتوكوندريا مقارنة مع sonicated عي...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

عزل الميتوكوندريا من كميات الحد الأدنى من الفأر العضلات الهيكلية للإنتاجية عالية صفيحة ميكروسكوبية الجهاز التنفسي القياسات

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K Views.  Virginia Tech. Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Video: Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

الكبار والعضلات والهيكل العظمي الجنينية Microexplant الثقافة وعزل خلايا جذعية عضلية هيكلية

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

تنقية الأسلاف من الهيكل العظمي والعضلات

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

العزلة من الدم والأوعية المشتقة متعددة القدرات السلائف من العضلات الهيكلية البشرية

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

الإنتاجية العالية للكشف عن المغيرون الكيميائية من الجينات المشاركة ما بعد transcriptionally الخاضعة للرقابة

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

عزل الميتوكوندريا سليمة من العضلات الهيكلية التي كتبها التفاضلية الطرد المركزي لقياسات عالية الدقة قياس التنفس

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

عزل وتوصيف الإكسوسومات من الخلايا الليفية العضلية الهيكلية

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

عزل الخلايا العضلية الأذينية والبطينية عالية الجودة للقياسات المتزامنة لتيار الكالسيوم2+ العابر وتيار الكالسيوم من النوع L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

تحضير تجانس عضلات الهيكل العظمي للفئران لقياسات النترات والنتريت

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...