Name: isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells
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وأيد هذا العمل من قبل المعاهد الوطنية للصحة / المعهد الوطني للاضطرابات العصبية والسكتة الدماغية (NIH / NINDS) يمنح R01-NS074387، R01-R21 NS057711 و-NS091555 إلى MGC. NIH / NINDS منح R01-NS061107، R01-NS076991، R01-R21 NS082311 و-NS084275 لااا. المنح المقدمة من سعيد قلوب ليا، جامعة ميتشيغان مركز السرطان الشامل تمنح للMGC وPRL؛ قسم جراحة المخ والأعصاب، جامعة ميشيغان كلية الطب. معهد ميشيغان لالسريرية والبحوث الصحية، بدعم من المعاهد الوطنية للصحة منحة 2UL1-TR000433. جامعة ميشيغان السرطان غرانت الأحياء التدريب بدعم من المعاهد الوطنية للصحة / NCI (المعهد الوطني للسرطان) منحة T32-CA009676؛ جامعة ميشيغان التدريب في الأساسية والسريرية علم الأعصاب بدعم من المعاهد الوطنية للصحة / NINDS منح T32-NS007222. وجامعة ميشيغان برنامج التدريب العلماء الطبية بدعم من المعاهد الوطنية للصحة / NIGMS (المعهد الوطني لعلوم الطب العام) منح T32-GM007863. المؤلفونإعادة شاكرين للقيادة الأكاديمية والدعم الذي تلقاه من الدكتور كارين Muraszko وقسم الجراحة العصبية. لM. داهلغرن، D. TOMFORD، وS. نابوليتان للدعم الإداري رائع. لM. Dzaman للحصول على المساعدة الفنية المعلقة. وفيل F. جينكينز للحصول على الدعم السخي تجاه شراء زايس 3D مجهر المسح الإلكتروني. علينا أيضا أن نعترف المختبر Kuchroo في كلية الطب بجامعة هارفارد الذي تم وضع نسخة معدلة من استراتيجية بوساطة وسائل الإعلام الكثافة الطرد المركزي لعزل الخلايا وحيدة النواة الدماغ.

ملاحظة: يرجى مراجعة بروتوكول كامل قبل إجراء التجارب. يجب الحصول على موافقة لاستخدام الحيوانات الفقارية من اللجنة المؤسسية المناسبة على استخدام ورعاية الحيوانات قبل المتابعة. 1. إعداد الخلايا السرطانية لداخل الجمجمة Engraftment <ol styl...

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يصف هذا البروتوكول وسيلة قوية وقابلة للتكرار للعزلة وتدفق cytometric تحليل PBMCs التي تسللت الماوس في وقت مبكر ورم في المخ المكروية. يتم إنشاؤها تعليق خلية الورم في تركيز يحددها التجريبي التي المغروسة stereotactically في المخطط من مخ الفأر. ثم يصرح باستخدامها الفئران في نقطة زمنية ...

الاورام الدبقية هي فئة من سرطانات الدماغ الظهارية العصبية الناجمة عن الدبقية تتحول داخل الجهاز العصبي المركزي (CNS). جميع الاورام الدبقية، ومنظمة الصحة العالمية (WHO) الصف الرابع الورم، أو ورم أرومي (GBM)، هو الأكثر شيوعا وفتكا 1. GBM هو صهر للغاية لرعاية المعايير من الحالية التي تتكون من الورم استئصال إلى أقصى حد ممكن تليها الأشعة بالإضافة إلى العلاج الكيميائي وما يصاحبه من المواد المساعدة مع temozolomide 2. هذه السرطانات القاتلة تحمل أحوال الطقس الكئيب أشهر فقط 15-18 من البقاء على قيد الحياة من وقت التشخيص الأولي مع 5٪ فقط من المرضى على قيد الحياة المرض بعد 5 سنوات 3. وجود حاجز الدم في الدماغ (BBB)، أدت عدم وجود خلايا مقدمة للمستضد المهنية (ناقلات الجنود المدرعة)، ووجود مجهولون في وقت سابق من الهياكل اللمفاوية الحسنة النية داخل الدماغ 4 لمفهوم GBM حظا المناعة كما. ومع ذلك، العديد من الدراسات SHO الآن<html> ث أن هذه السرطانات الدماغ في الواقع يولد تجنيد الخلايا المناعية الطرفية التي هي في الغالب النخاعي في الأصل والتي تشمل حيدات، الضامة، والخلايا القامع المستمدة النخاعي (MDSCs) 5. كما يؤثر GBM نشاط المقيمين في الدماغ الدبقية الصغيرة لتصبح مؤيدة للمكون للأورام 6،7. الخلايا اللمفاوية مثل خلايا CD8 + T 8 و CD56 + الخلايا القاتلة الطبيعية 9 موجودة أيضا داخل المكروية الورم، لكن بأعداد أقل بكثير، وهذه حقيقة يعتقد أن يكون راجعا إلى وظيفة المثبطة للمناعة بتحريض من العوامل المستمدة من الورم في ورم المرتبطة الضامة ( TAMS) 10. خلايا CD4 + T موجودة أيضا في GBM، ولكن الكثير من هذه الفئة من السكان تعرب عن قلقها أيضا CD25 وFoxP3، صناع مناعة T التنظيمي (T ريج) خلايا (11). الدولة المثبطة للمناعة العامة للGBM يتوج في تعزيز الهروب المناعية والأورام تقدم 12. = &quot;jove_content&quot;&gt; فهم أفضل للآليات GBM المناعة أمر بالغ الأهمية لوضع استراتيجيات فعالة immunotherapeutic تهدف إلى تحفيز الجهاز المناعي ضد الورم. على مدى السنوات ال 15 الماضية عملت مختبرنا للتغلب على آليات الدماغ ورم immunosuppresson من أجل تطوير فعال immunotherapeutics جديدة لمكافحة GBM 13-19. وقد أدى تتويجا لهذا العمل الآن لتجربة سريرية تهدف إلى تقييم السامة للخلايا جنبا إلى جنب والعلاجية المناعة تنشيطية لمرضى شخصت حديثا GBM (ClinicalTrials.gov معرف: NCT01811992). يظهر عملنا الأحدث التي GL26 الماوس والفئران CNS-1 خلايا GBM كتلة مضادة للورم الخلايا NK المراقبة المناعي عن طريق إنتاج كميات كبيرة من الربط β-غالاكتوزيد-كتين galectin-1 (غال-1) 20. وقد تجلى ذلك من خلال قمع التعبير عن غال-1 في خلايا الورم باستخدام shRNA بوساطة ضربة قاضية الجينات. في المختبر التجربهوأظهرت البحوث أن خلايا الورم غال 1-نقص انتشرت عادة في الثقافة، ولكن خضعت الرفض السريع قريبا بعد engraftment داخل الجمجمة في مسانج C57BL / 6J أو RAG1 - / - الفئران، وبالتالي تأسيس استقلال T- أو B- الخلايا على هذا الشكل من رفض الورم. NK immunodepletion الخلايا مع anti-asialo GM 1 المصل المضاد أو الأجسام المضادة وحيدة النسيلة NK1.1 أدى إلى الاستعادة الكاملة لنمو الورم داخل القحف غال-1-نقص، وإنشاء دور الخلايا القاتلة الطبيعية في غال-1-نقص رفض الورم. وتبين لنا الآن أن immunodepletion من GR-1 + / CD11b + خلايا الدم النخاعي غير كافية لمنع غال-1-نقص رفض الورم على الرغم من وجود خلايا NK، مما يكشف عن دور مساعد لا غنى عنه لخلايا الدم النخاعي في العون من غال NK بوساطة تحلل الورم -1-ناقص (بيانات غير منشورة). وأدت هذه النتيجة غير متوقعة لنا لوضع بروتوكول شامل لعزل وتحليل خلايا الدم وحيدات النوى المحيطية (PBMCs) أنالتسلل المكروية ورم في المخ بعد وقت قصير من engraftment داخل الجمجمة حتى يتسنى لنا أن تميز أفضل الأحداث تسلل المناعية التي المسند غال-1-نقص رفض الورم. ويتجلى الأسلوب هنا باستخدام خلايا الورم الماوس GL26 أن mCitrine صريحة جوهري البروتين الفلوري، ودعا GL26-سيت، والتي تسمح المباشر التصور الخلايا السرطانية بواسطة المجهر مضان 21. وهذه الخلايا المغروسة stereotactically في الدماغ من مسانج C57BL / 6J الفئران ويسمح لتنمو لمدة 24، 48، أو 72 ساعة قبل الماوس القتل الرحيم. ثم يتم عزل PBMCs-التسلل الورم وimmunolabeled استخدام الألغام المضادة -CD45، -Gr-1، -CD11b و-NK1.1 الأجسام المضادة سطح الخلية مع immunolabeling داخل الخلايا لجرانزيم B (GzmB). هذه تركيبة معينة من الأجسام المضادة يسمح لتحديد-التسلل الورم GR-1 + / + CD11b خلايا الدم النخاعي وNK1.1 +، وخلايا NK، أنواع الخلايا لدينا بالتابعين المتورطين في رفض الورم غال-1-ناقص. ملف تسلل المناعي للغال-1-نقص GL26-سيت الورم، ويشار إليها هنا ب GL26-سيت-gal1i، ثم يتم مقارنة بما كان عليه من الاورام الدبقية التعبير عن المستويات العادية من غال-1 يسمى GL26-سيت-NT التي تحتوي على عدم تستهدف السيطرة shRNA القاسي. بروتوكول يبدأ مع الوصف على كيفية الثقافة الخلايا GL26-سيت الورم في المختبر، والتي تبعتها شرحا عن كيفية تدبر orthotopically هذه الخلايا في الجسم المخطط من مسانج C57BL / 6J الفئران. ومن ثم تمضي إلى تعداد الخطوات المتبعة في عزلة وimmunolabeling من PBMCs-التسلل الورم لتحليل تدفق cytometric. ويخلص البروتوكول مع شرح لتحليل البيانات القياسية وتمثيل رسومي. تكشف المظاهرة أن كلا من GR-1 + / + CD11b خلايا الدم النخاعي وخلايا NK1.1 + NK تتراكم تفضيلي ضمن المكروية ورم في المخ غال-1-نقص في غضون 48ساعة من زرع الورم، ونتيجة لذلك مما يساعد على تفسير سبب هذه الأورام الخضوع بسرعة كامل الورم تحلل حوالي 1 أسبوع engraftment بعد ورم-20. هذه الطريقة قابلة للتكيف بسهولة إلى عدد من مختلف في الجسم الحي التصاميم التجريبية التي تتطلب بيانات الزمنية على تسلل المناعة في الدماغ. A التجريبي واحد يمكن أن تؤدي البروتوكول من حصاد الدماغ لتحليل تدفق cytometric من PBMCs-التسلل الورم في حوالي 4-6 ساعة اعتمادا على عدد من العينات لتحليلها. ويمكن أيضا طريقة دمجه مع التجارب التي تهدف إلى تميز الملف الشخصى تعميم PBMCs في الفئران الحاملة للورم للمقارنة مع تلك التي تتسلل إلى الدماغ وذلك لتحديد الظواهر كبت المناعة بفعل تحديدا المكروية الورم. تطبيق هذا وما شابهه من الأساليب ينبغي أن تيسر فهم أفضل للعوامل متورطة في تهريب الخلايا المناعية الطرفية في المكروية ورم في المخ.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td>without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm deep</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% NaCl injection, USP</td>
			<td>Hospira</td>
			<td>NDC: 0409-4888</td>
			<td>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride injection</td>
			<td>Orion Pharma</td>
			<td>NDC: 52483-6298</td>
			<td>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clippers</td>
			<td>3M</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone-iodine solution</td>
			<td>Aplicare</td>
			<td>82-217</td>
			<td>NDC: 52380-1905</td>
		</tr>
		<tr>
			<td>Sterile petrolatum ophthalmic ointment&#160;</td>
			<td>Dechra</td>
			<td>NDC: 17033-211</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol prep pads</td>
			<td>Kendall</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td>non-woven, 4&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml conical polypropylene microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse stereotactic frame</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Philips Burton</td>
			<td>CS316W</td>
			<td>Coolspot II Variable Spotlight</td>
		</tr>
		<tr>
			<td>Curved dissecting forceps</td>
			<td>Ted Pella Inc.</td>
			<td>5431</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri retractors&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</td>
			<td>1100-01</td>
			<td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td>
		</tr>
		<tr>
			<td>Engraving Cutter</td>
			<td>Dremel</td>
			<td>105</td>
			<td>1/32&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L glass screw-cap storage bottle</td>
			<td>Corning</td>
			<td>1395</td>
			<td>Type 1, Class A borosilicate glass</td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/ml solution</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole Parmer Instruments Group</td>
			<td>77200-60</td>
			<td>Master Flex Easy Load II console drive</td>
		</tr>
		<tr>
			<td>compressed carbogen (95% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>from Clostridium histolyticum&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Antistatic polystyrene hexagonal weighing dishes</td>
			<td>Ted Pella Inc.</td>
			<td>20157-3</td>
			<td>top I.D. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</td>
			<td>Garvey</td>
			<td>40475</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone rongeurs</td>
			<td>FST</td>
			<td>16001-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13014-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers&#160;</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td>
			<td>Biolegend</td>
			<td>103128</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td>
			<td>eBiosciences&#160;</td>
			<td>17-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td>
			<td>BD Pharmigen</td>
			<td>553128</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control</td>
			<td>Biolegend</td>
			<td>400632</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Special Order Research Product flow cytometer/cell sorter</td>
			<td>BD&#160;</td>
			<td>650033</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG603</td>
			<td>model: SterilGARD III Advance</td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

وقد أظهرت مختبرنا مؤخرا أن القاتلة الطبيعية (NK) الخلايا قادرة على القضاء على زرع orthotopically GL26 الماوس والفئران CNS-1 الاورام الدبقية الخبيثة قريبا بعد engraftment داخل الجمجمة إذا أديت الخلايا السرطانية نقص في التعبير عنها من كتين galectin β-غالاكتوزيد ملزم -1 (غال-1). مزيد من العمل مؤخرا يقول إن عدد سكانها GR-1 + / + CD11b خلايا الدم النخاعي أمر بالغ الأهمية لهذا الغرض. لفهم أفضل للآليات التي NK وخلايا الدم النخاعي التعاون لمنح رفض الورم غال-1-نقص قمنا بتطوير بروتوكول شامل لعزل وتحليل خلايا الدم وحيدات النوى الطرفية-التسلل الورم (PBMC). ويتجلى الأسلوب هنا بمقارنة PBMC تسلل إلى المكروية الورم من غال-1، معربا عن GL26 الاورام الدبقية مع أولئك الذين أصبحوا غال-1-ناقص عبر shRNA ضربة قاضية. بروتوكول يبدأ مع وصفا لكيفية الثقافة وإعداد GL26 مLLS للتلقيح في مسانج C57BL / 6J مخ الفأر. ثم يوضح الخطوات المتبعة في عزلة وتدفق تحليل cytometric من PBMCs-التسلل الورم من أوائل المكروية ورم في المخ. هذه الطريقة قابلة للتكيف مع عدد من التصاميم التجريبية في الجسم الحي التي تتطلب بيانات الزمنية على تسلل المناعة في الدماغ. طريقة حساسة وقابلة للتكرار للغاية، كما PBMCs-التسلل الورم يمكن عزله عن الأورام داخل الجمجمة في أقرب وقت 24 ساعة بعد ورم engraftment مع عدد خلايا مماثلة لوحظ من وجهة يقابل الأورام وقت خلال تجارب مستقلة. A التجريبي واحد يمكن أن تؤدي الأسلوب من حصاد الدماغ لتحليل تدفق cytometric من PBMCs-التسلل الورم في ما يقرب من 4-6 ساعة اعتمادا على عدد من العينات لتحليلها. كما يمكن استخدام نماذج الورم البديلة و / أو الأجسام المضادة الكشف عن خلية محددة وفقا لتقدير التجريبيون على تقييم تسلل العديد من إمان أخرىأنواع الخلايا (ه) من الفائدة من دون الحاجة لإجراء تعديلات على الإجراء العام.

يتم استخدام استراتيجية النابضة التالية لتجربة نموذجية: FSC-A مقابل SSC-A → SSC-H مقابل SSC-W → FSC-H مقابل FSC-W → CD45 مقابل العد → GR-1 مقابل CD11b → NK1.1 مقابل العد. وضعت بوابات على GR-1 + / + CD11b خلايا الدم النخاعي وخلايا NK1.1 + NK ثم يتم الطبقية على أساس GzmB التعبير (الشكل...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

المقدمة هنا هي طريقة واضحة لعزل وتدفق تحليل cytometric من الخلايا وحيدة النواة الدم المحيطي-التسلل الورم يمكن أن ينتج تعتمد على الوقت بيانات كمية عن وضع عدد وتنشيط الخلايا المناعية التي تدخل المبكر المكروية ورم في المخ.

العزلة وتحليل تدفق Cytometric من-التسلل الورم خلايا الدم المحيطي وحيدة النواة

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

عزل خلايا فأر التجويف البريتوني

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

كشف الجليكوجين في خلايا الدم المحيطي وحيدات النوى مع الدوري حمض شيف تلطيخ

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

العزلة وتحليل تدفق Cytometric من الخلايا المناعية من الدماغ الدماغية ماوس

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

تدفق تحليل Cytometric من القاتل الطبيعي خلية التحللي آخر في الدم الجامع البشري

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

العزلة وتدفق Cytometric تحليل الإنسان باطن عنق الرحم غاما دلتا الخلايا التائية

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

الهضم الكبد مورين لتحليل تدفق سيتوميتريك اللمفاوية خلايا غشائي

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

هضم عيون الماوس كله لتحليل Cytometric تدفق متعدد المعلمة من البلاعات أحادية النوى

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

عزل الخلايا التائية القائمة على التعويم وتنشيطها وتوسيعها من عينات خلايا الدم أحادية النواة المحيطية البشرية باستخدام الفقاعات الدقيقة

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

مجموعة PBMC والتخزين بالتبريد لتحليل الطاقة الحيوية للميتوكوندريا

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

تسليط الضوء على المؤلف: الحفاظ على معلمات الطاقة الحيوية في خلايا الدم أحادية النواة المحيطية بعد الحفظ بالتبريد 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

الحفظ بالتبريد وتقييم الطاقة الحيوية لخلايا الدم البشرية أحادية النواة المحيطية

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...