Name: imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma spheroids
Uploaded: 2015-12-28T14:00:00
Description: Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).

1. FUCCI تنبيغ والثقافة خلية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> FUCCI تنبيغ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء خطوط الخلايا معربا عن ستابلي FUCCI يبني mKO2-hCdt1 (30-120) وMAG-hGem (1-100) 15 باستخدام الفيروسة البطيئة المشارك تنبيغ كما هو موضح سابقا <su...

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حدد تحليل الصور الآلي شبه والداخلية G1 القبض المنطقة كروي، وتنتشر الطبقات الخارجية. هذه الطريقة يمكن أن تستخدم في الكروية الحية باستخدام الجزء البصري، أو في أقسام كروي ثابت، لتحديد التغييرات ليس فقط في دورة الخلية ولكن علامة التعبير (عن طريق المناعي)، موت الخلايا، أو...

من المعروف أن الكروية متعددة الخلايا 3D كنموذج الورم لعدة عقود، إلا أنه في الآونة الأخيرة فقط أنهم جاءوا إلى استخدام أكثر شيوعا نموذجا في المختبر لكثير من السرطانات الصلبة. ويتزايد استخدامها في الإنتاجية العالية شاشات اكتشاف الأدوية كمادة وسيطة بين المجمع ومكلفة والنماذج الحية تستغرق وقتا طويلا وبسيط، وانخفاض تكلفة 2D نموذج أحادي الطبقة 1-6. دراسات في الثقافة 2D غالبا ما تكون غير قادرة على تكرارها في الجسم الحي. نماذج كروي من أنواع عديدة من السرطان قادرة على محاكاة خصائص النمو، والحساسية المخدرات، واختراق المخدرات، والتفاعلات خلية خلية، وتوافر المقيد من الأوكسجين والمواد المغذية وتطوير نخر أن ينظر في الجسم الحي في الأورام الصلبة 6-11. الكروية تطوير نواة نخرية، وهي منطقة هادئة أو G1 القبض المحيطة الأساسية، والخلايا المتكاثرة في محيط كروي 7. تطوير هذه المناطققد تختلف تبعا لكثافة الخلايا، ومعدل انتشار وحجم كروي 12. تم الافتراض بأن عدم التجانس الخلوية ينظر في هذه المناطق الفرعية المختلفة قد يسهم في علاج السرطان المقاومة 13،14. وبالتالي القدرة على تحليل الخلايا في هذه المناطق أمر حاسم لردود المخدرات فهم الورم بشكل منفصل. ويستند (FUCCI) نظام مضان ubiquitination مؤشر دورة الخلية على أحمر (Kusabira أورانج - KO) والأخضر (الأعظمي الأخضر - AG) العلامات الفلورية من Cdt1 وgeminin، والتي تدهورت في مراحل مختلفة من دورة الخلية 15. وبالتالي نواة الخلية تظهر حمراء في G1، الأصفر في أوائل S والخضراء في S / G2 / M المرحلة. وصفنا هنا طريقتين التكميلية على حد سواء باستخدام FUCCI لتحديد دورة الخلية، جنبا إلى جنب مع استخدام برامج التصوير أو تدفق نشر صبغة الخلوي فحص لتحديد ما إذا كانت الخلايا الموجودة في مركز اعتقال G1 أو proliferatin الخارجيز حلقة، والمسافة من الخلايا الفردية من على حافة كروي. وقد وضعت هذه الأساليب لدينا في المنشور السابق، حيث أثبتنا أن خلايا سرطان الجلد في مناطق ميتة في وسط كروي و / أو في وجود العلاجات المستهدفة تكون قادرة على البقاء في اعتقال G1 لفترات طويلة من الزمن، ويمكن إعادة دخول دورة الخلية عندما تنشأ ظروف أكثر ملاءمة 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>agarose low melting point</td>
			<td>Life Technologies</td>
			<td>16520-050</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>noble agar&#160;</td>
			<td>Sigma</td>
			<td>A5431</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>agarose for spheroids</td>
			<td>Fisher Scientific</td>
			<td>BP1356-100</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>0.05% trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin</td>
			<td>Sigma</td>
			<td>HT5014-1CS</td>
			<td>CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do &#160;not breath fumes (open in a fume cupboard).</td>
		</tr>
		<tr>
			<td>live/dead near IR</td>
			<td>Life Technologies</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>vibratome</td>
			<td>Technical Products International, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>coulture cup</td>
			<td>Thermo-Fisher Scientific</td>
			<td>SIE936</td>
			<td>Mold for sectioning spheroids</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Invitro</td>
			<td>FAL353072</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Sigma</td>
			<td>C5138&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer analyser</td>
			<td>Becton Dickinson</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>flowjo</td>
			<td>Tree Star</td>
			<td></td>
			<td>Flow cytometry software</td>
		</tr>
		<tr>
			<td>Vaccuum grease</td>
			<td>Sigma</td>
			<td>Z273554</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FUCCI (commercial constructs)</td>
			<td>Life Technologies</td>
			<td>P36238</td>
			<td>Transient transfection only</td>
		</tr>
		<tr>
			<td>Cell strainer 70 um</td>
			<td>In Vitro</td>
			<td>FAL352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (sterile)</td>
			<td>In Vitro</td>
			<td>FAL352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (non-sterile)</td>
			<td>In Vitro</td>
			<td>FAL352008</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

هناك عدة طرق لإنتاج الأجسام الشبه الكروية الورم، ويستخدم هذا البروتوكول طريقة نمو غير ملتصقة، حيث يتم استزراع الخلايا على أجار أو 3،7،9 الاغاروز الشكل يظهر 1 مثال على كروي C8161 سرطان الجلد بعد 3 أيام على أجار. الكروية C8161 شكل الكروية العادية الحج?...

Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

تحليل يستند الخلوي-Imaging- وتدفق الوظيفة خلية ودورة الخلية في 3D الميلانوما الأجسام الشبه الكروية

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

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Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

زراعة الخلايا المشتقة المريض وعزل CD133<sup&gt; +</sup&gt; خلايا الجذعية السرطانية المزعومين من الميلانوما

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

الإنتاجية العالية التدفق الخلوي القائم على الفحص لكشف خلية أضداد N-ميثيل مد اسبارتاتي مستقبلات الدوبامين-2 أو مستقبلات في مصل الإنسان

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

روز البنغال Photothrombosis بواسطة التصوير الضوئي متحد البؤر<em&gt; في فيفو</em&gt;: نموذج لسفينة واحدة السكتة الدماغية

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

فحص الخلوي على أساس تدفق لقياس إمكانات غشاء الميتوكوندريا في Myocytes القلب بعد نقص/ريوكسيجينيشن

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

تحليل كمي [18F]-NAF-PET-MRI لتقييم دوران العظام الديناميكية في مريض يعاني من آلام الظهر المنخفضة في Facetogenic

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

تحليل تدفق الخلايا الخلوية من المجموعات الفرعية الخلية المناعية داخل الطحال مورين, نخاع العظام, الغدد الليمفاوية والأنسجة الزليلي في نموذج هشاشة العظام