Эта работа была поддержана грантами национального здравоохранения и медицинских исследований Совета Австралии (NHMRC) (526615 и 545906) и частично OIS Программа викторианской правительства. DS является членом NHMRC.

1. Подготовка [3 H]-холестерина <ol><li> В вытяжной шкаф, отказаться от требуемого количества [3 H] холестерина в 1,5 мл микроцентрифужную трубки (0,5 мКи (19 кБк) в каждую лунку необходимо для типичного анализа). </li><li> Если [3 H]-холестерина приостановлена ​​в толуоле, высушите его полностью с N2 газ и ресуспендируют со 100%-ным этанолом до конечной концентрации 1 мкКи (37 кБк / мкл. Vortex и хорошо перемешать. </li></ol> 2. Покрытие клетки и маркировки сотовых Холестерин Этот протокол был протестирован с помощью следующих типов клеток: человеческие моноциты 4,5, THP-1 человеческих моноцитов-макрофагов 6,7,8, RAW 264.7 мышиных макрофагов 5,9,10,11, HeLa клетки 12 человек эндотелия пупочной вены клеткам (HUVEC), ВНК-21 клеток 9, человеческих и мышиных фибробластов 13,14, HepG2 клетках человека гепатокарциномы 15 и в измененном виде, из тромбоцитов &lt;sup&gt; 16. <ol><li> Ресуспендируйте клеток и их сосчитать. Пластина клеток в 12-луночных планшетов на конечной плотности 0.2x10 6 клеток на лунку в 0,9 мл полной среды. Клетки будут продолжать расти и достигнет 0.8x10 6 клеток на хорошо ко времени истечения эксперимента. Для клеток, которые не делятся, например, дифференцированный ТНР-1 клеток, плотности посева должна быть 0.8x10 6 клеток на лунку. При использовании 6 - или 24-луночный, двойных или половину числа клеток на лунку соответственно. Количество скважин должна быть достаточной для определения четырех экземплярах для каждой экспериментальной состоянии. </li><li> Если дифференциация ТНР-1 клеток необходимо, добавьте PMA (конечная концентрация 0,1 мкг / мл) в средствах массовой информации в течение 48-72 часов. </li><li> Добавить небольшую аликвоту СМИ в отцентрифугировать пробирку с [3 H] холестерина. </li><li> Вымойте стороны трубки со средствами массовой информации, прежде чем добавлять [3 H] холестерина в отдельных аликвоту полный СМИ (100 мкл / лунку). </li> &lt;LI&gt; Добавить средах, содержащих [3 H] холестерина в лунки с клетками (конечный объем на одну скважину составляет 1 мл). При отсутствии лечения клеток необходимо до маркировки, клетки могут быть покрыты в [3 H] холестерина содержащей среде. </li><li> Инкубируйте клетки в течение 48 часов в культуре клеток инкубатор (37 ° C, 5% CO 2). </li></ol> 3. Уравновешивание Инкубационный <ol><li> После 48 часов инкубации, проверьте клеток под микроскопом. Убедитесь, что они здоровы и находятся в примерно 80% слияния. </li><li> Удалить средах, содержащих [3 H] холестерина. Промойте клетки осторожно PBS. Повторите 3 раза стирки. </li><li> Подготовить бессывороточной СМИ. При необходимости активировать клетки, добавив LXR агониста (например, TO-901 317 на 1-4 ммоль / л) или цАМФ (0,3 ммоль / л, для клеток мышей происхождения только) в сыворотке крови свободной информации. </li><li> Добавить 500 мкл сыворотки свободных средств массовой информации в каждую лунку. </li><li> Выдержите в течение 18 часов в культуре клеток инкубатор (37 ° C, 5% CO 2). </li></ol> &lt;с классом = &quot;jove_title&quot;&gt; 4. Холестерин Инкубационный истечение <ol><li> После 18 часов инкубации в бессывороточной среде, проверьте клеток под микроскопом. Убедитесь, что они здоровы и сливной (минимум 80% слияния). </li><li> Подготовить решение холестерина истечения акцепторов в бессывороточной среде. Примерами акцепторов включают в себя: Аполипопротеина AI (апо-I) (конечная концентрация 10 мкг / мл) Липопротеинов высокой плотности (ЛПВП) (конечная концентрация 20 мкг / мл) Циклодекстрин (конечная концентрация 200 мкг / мл) Плазменные (конечная концентрация 1-2%) </li><li> Промойте клетки осторожно PBS. </li><li> Добавить 250 мкл сыворотки среде с акцепторами в каждую лунку. </li><li> Оставьте один набор скважин для определения фоне оттока (отток в сыворотке крови свободных средств массовой информации с NO акцептор) </li><li> Инкубируйте клетки в течение 2 часов в культуре клеток инкубатор (37 ° C, 5% CO 2). Срок истечения инкубационного может варьироваться от 30 минут до 8 часов, если требуется. </li></ol> 5. Обработка Samples <ol><li> Через 2 часа инкубации проверить клетки под микроскопом. </li><li> Сбор СМИ в пробирки на 1,5 мл отцентрифугировать. Спиновая при 14000 оборотов в минуту в течение 1-10 минут при комнатной температуре, чтобы удалить продукты распада клеток. </li><li> Передача 100 мкл среды на 7 флакон сцинтилляционных мл. Добавьте 5 мл Insta-гель Plus (PerkinElmer) и вихревой смеси. Хранить оставшихся образцов при температуре 4 ° C. </li><li> Установите плиты в морозилку на 30 минут. Добавить 500 мкл дН 2 O в каждую лунку. Проверьте клетки под микроскопом, чтобы убедиться, что все клетки подняли со дна скважины. Если все же сторонник, либо оставить пластины с дН 2 O при температуре 4 ° С в течение ночи или царапина скважин. </li><li> После того как все клетки сняты, пипетка вверх и вниз, чтобы разбить ячейку сгустки. Передача 100 мкл на 7 флаконов мл сцинтилляционного. Добавьте 5 мл Insta-гель Plus (PerkinElmer) и вихревой смеси. Храните оставшиеся пластины при температуре 4 ° C. </li><li> Поместите сцинтилляционных флаконов на сцинтилляционных счетчиков. Настройка счетчика рассчитывать на [3 H] вDPM единиц. </li></ol> 6. Анализ результатов <ol><li> Уровень холестерина истечения обычно выражается в виде доли от холестерина переехал из клетки акцептора. Используется следующая формула: </li></ol><img alt="Уравнение 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> Конкретные истечения рассчитывается как разница между истечения в присутствии или отсутствии акцептора (пустой). </li></ol><img alt="Уравнение 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Хронология <img alt="Рисунок 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Представитель Результаты Пример результатов эксперимента холестерина истечения показано на рис. 1. В этом эксперименте THP-1 человека моноциты дифференцируются в макрофаги и холестерина истечение различных акцепторовиспытания. Холестерин истечения в среде с не акцепторами (&quot;пустой&quot;) было 0,79%, и это значение рассматривается как неспецифический истечения и вычитается из других ценностей. Холестерин истечения для человеческого апо-I (конечная концентрация 30 мкг / мл) 4,75%. Образец ссылки плазма была включена в этот эксперимент, чтобы контролировать между экспериментальной изменчивости. Эталонного образца могут быть использованы для нормализации данных между большим числом экспериментов, но мы сочли разумным, чтобы повторить тест, если изменчивость высока. Пациент плазмы (конечная концентрация 2%) был испытан до и после пациент лечился с помощью лекарств. Сделан вывод, что у этого пациента препарат оказывает негативное воздействие на способность плазмы поддерживать холестерин истечения. Другой пример результате истечения эксперимента приведена на рис. 2. В этом эксперименте RAW 264.7 макрофаги активирован или не активирован ночной инкубации с LXR агониста TO-901 317 (конечная концентрация1 мкмоль / л) и холестерина истечения в том же образце плазмы крови человека (2%) была проверена. Был сделан вывод, что активация клеточного выражение перевозчиков ABC с агонистов LXR увеличивает пропускную способность клеток к выпуску холестерина внеклеточной акцептором. <img alt="Рисунок 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Рисунок 1. Холестерин истечение из ТНР-1 клеток различных акцепторов. Процент холестерина истечения (т.е. доля меченых холестерин перемещается из клеток к указанной акцептор) показана после вычитания из пустых значений. Среднее ± стандартное отклонение в четырех экземплярах определений, * р &lt;0,05. <img alt="Рисунок 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Рисунок 2. Холестерин истечение из RAW 264.7 активирован или не активирован с LXR агониста плазмы крови человека. Процент холестерина истечения (т.е. доля меченых холестерина переехал из клеткиуказанные акцептор) показана после вычитания пустые значения. Среднее ± стандартное отклонение в четырех экземплярах определений, * р &lt;0,001.

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Авторы заявляют нет конфликта интересов, связанных с этим исследований.

Описанный метод измерения холестерина истечения предназначен для измерения движения холестерина из клеток внеклеточную акцептором холестерина. Есть несколько важных соображений, в понимании этой методологии. Маркировка и равновесия В описанной методо?...

<table border="1"><tbody><tr><td> Реагенты \ Клетки </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> ВНК-21 </td><td> HFF </td></tr><tr><td> Полное СМИ </td><td colspan="3"> RPMI - 10% FCS - 1% L-глутамина - 0,2% Pen стрептококк </td><td> M199 - 5% FCS - 1% L-глутамина - 1% Pen стрептококк - 2% HEPES (1M) - 7% эндотелиальных дополнение рост клеток (ЭКГ) </td><td colspan="2"> DMEM - 10% FCS - 1% L-глутамина - 0,2% Pen стрептококк </td></tr><tr><td> Сыворотка свободные СМИ </td><td colspan="3"> RPMI - 1% L-глутамина - 0,2% Pen стрептококк </td><td> M199 - 1% L-глутамина - 1% Pen стрептококк - 2% HEPES (1M) - 7% эндотелиальных клеток растиго дополнения (ЭКГ) </td><td colspan="2"> DMEM - 1% L-глутамина - 0,2% Pen стрептококк </td></tr><tr><td> Прохождение метод </td><td> 1 х трипсина </td><td> Приостановление </td><td> Скоблить </td><td colspan="3"> 1 х трипсина </td></tr><tr><td> Прохождение соотношение </td><td> 3:10 </td><td> 1:03 </td><td></td><td> 1:20 </td><td> 1:03 </td><td> 1:03 </td></tr><tr><td> Сотовые активации (опционально) </td><td></td><td colspan="5"> К-901 317 - LXR-агонистов - Окончательный конц. 4 мкМ </td></tr><tr><td> Другие реагенты </td><td></td><td> форбол 12-миристат-13-ацетат (PMA) - Дифференциация клеток - Окончательный конц. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

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Анализ оттока холестерина | Медицина | Видео

Cholesterol Efflux Assay

Холестерин анализ предназначен для количественного определения скорости оттока холестерина из культивируемых клеток и способность плазмы акцепторов принять холестерина выпустили из клеток. Анализ состоит из маркировки клеток холестерина, холестерина равновесия между внутриклеточными бассейны и выпуск холестерина внеклеточной акцептором.

Холестерин Анализ истечения

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29.5K Views.  Baker IDI Heart and Diabetes Institute. Холестерин анализ предназначен для количественного определения скорости оттока холестерина из культивируемых клеток и способность плазмы акцепторов принять холестерина выпустили из клеток. Анализ состоит из маркировки клеток холестерина, холестерина рав...

Video: Cholesterol Efflux Assay

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Экспериментальные Пробирной метастазы

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

В Vivo собак Пробирной функции мышц

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Мышь Модель Пробирной Роговица Карман для ангиогенеза исследование

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Сфероид Пробирной для измерения TGF-β-индуцированной Вторжение

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; В естественных условиях</em&gt; Анализ для тестирования проницаемости кровеносных сосудов

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; В ово</em&gt; CAM-анализа, как Xenograftmodel саркомы

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Имитация поджелудочной Нейропластичность:<em&gt; Экстракорпоральное</em&gt; Dual-нейрон Пластичность Анализ

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Трехмерная (3D) опухоли Сфероид Вторжение Пробирной

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Дифференциального воздействия гиполипидемические препараты в модуляции морфологией частиц холестерина

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.