Мы благодарим членов биологической лаборатории фагосомы за критическое прочтение рукописи. Эта работа поддержана грантом Гельмгольца Молодая следователь (инициатива и Сетевые средств из Гельмгольца) и Программы Priority SPP1580 Грант немецкого исследовательского совета (Deutsche Forschungsgemeinschaft, DFG).

<ol>
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Immunol. 14, 103-110 (2002).</li><li>Bohdanowicz, M., Cos&#237;o, G., Backer, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I+and+class+III+phosphoinositide+3-kinases+are+required+for+actin+polymerization+that+propels+phagosomes.">Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes.</a> J. Cell Biol. 191, 999-1012 (2010).</li><li>Hoffmann, E., 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=Initial+receptor-ligand+interactions+modulate+gene+expression+and+phagosomal+properties+during+both+early+and+late+stages+of+phagocytosis.">Initial receptor-ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis.</a> Eur. J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+phagocytic+engulfment+of+apoptotic+cells+by+macrophages+using+pHrodo+succinimidyl+ester.">Measurement of phagocytic engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.</a> Current protocols in immunology. 14, (2013).</li><li>Rogers, L., Foster, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+phagosomal+proteome+and+the+contribution+of+the+endoplasmic+reticulum.">The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 18520-18525 (2007).</li><li>Russell, D. G., Vanderven, B. C., Glennie, S., Mwandumba, H., Heyderman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+marches+on+its+phagosome:+dynamic+assays+of+phagosome+function.">The macrophage marches on its phagosome: dynamic assays of phagosome function.</a> Nat. Rev. Immunol. 9, 594-600 (2009).</li><li>Savina, A., Vargas, P., Guermonprez, P., Lennon, A. -. M., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pH,+ROS+production,+maturation,+and+degradation+in+dendritic+cell+phagosomes+using+cytofluorometry-based+assays.">Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays.</a> Methods. Mol. Biol. 595, 383-402 (2010).</li></ol>

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В следующем разделе мы обсудим важные этапы предложенного метода и его ограничения. Кроме того, мы подключим некоторые из распространенных проблем, с их решениями, ввести возможные изменения и рассмотреть преимущества нашего метода, а также подходящие дополнительные методы для этого ...

Профессиональные фагоциты, включая макрофаги, играют решающую роль в иммунной системе. Помимо того, что первая линия обороны в врожденной иммунной системы, они также играют важную роль в активации адаптивного иммунитета через их сигнализации и антигенпредставляющих роль 1-3. В то время как функция профессиональных фагоцитов многогранна, Фагосомы созревания является критическим основой бактерицидного и антигена функции обработки профессиональных фагоцитов 4,5. После опосредованного рецептором охвате и поглощения в фагоцитарной цели, такие как бактерии, возникающий Фагосомы проходит через сложную и хорошо организованной последовательности взаимодействия и обменов с отсеках эндоцитотического сети и нескольких других клеточных органелл 6. Характер соединений обменялись с этими клеточными лиц, а также регулирования и сроки событий слияния определяет просвета phagosomal среду и фагоМембрана состав OMAL и, таким образом, 7, судьба созревания фагосомы 8. Физиологическое значение фагосомы созревания иллюстрируется разнообразными стратегиями, используемыми различными внутриклеточных патогенов вырваться из, ареста или подорвать фагосомы созревание 9. Большинство из этих стратегий прямо или косвенно предотвратить окончательное и критическую стадию процесса созревания фагосомы: слияние фагосомы с конца эндосомный / лизосомальных отсеков, которые придают им основной массы гидролитических ферментов и антибактериальными факторов зрелой фагосомы 7 , 8,10. Анализ этой заключительной и важный шаг, следовательно, может предоставить нам сильных показателей о состоянии созревания фагосом и если естественное физиологическое состояние положительно или отрицательно быть затронуты в конкретных экспериментальных условиях, что в настоящее время используются. Покойный эндосомный / лизосомные отсекс, как правило, рассматривается как терминал отсеки эндоцитотического пути, определенные и отличающиеся от ранних эндоцитотических отсеков наличием различных, Стадия конкретных, молекулы-маркеры. Например гидролитических ферментов или мембранные компоненты, такие как Лизосомные связанных мембранных белков (ламп) среди других 11. Терминал эндоцитотический отсеки-отныне называют просто лизосом-также служить в качестве основного места для заключительных этапах пищеварения в конце фагоцитарной / эндоцитотический пути 12. Таким образом, неперевариваемые зонды могут быть загружены через эндоцитоз и макропиноцитозом из внеклеточной среды и транспортируется через эндоцитоза пути к лизосом, где она накапливается 13,14 после выполнения определенного цикла поглощения и охоты. Флуорофора, конъюгированным зонды для флуоресцентной микроскопии, такие как органического полимера неперевариваемые декстрана обычно используются в качестве endocyticprobes 15-17. <p clasS = &quot;jove_content&quot;&gt; В этом методе, описаны использование микросфер, покрытых IgG-фагоцитарной как груза для исследования фагосомы созревание путем анализа доставку контента лизосом просвета в фагосом. С помощью флуорофора-сопряженных декстрана зонд как легко выявляемого маркера лизосом в прямом эфире костного мозга, полученных макрофагов (BMM) и покадровой видео изображения с конфокальной лазерной сканирующей микроскопии, мы следуем динамический процесс доставки грузов в фагосом с высоким временное разрешение. Затем мы опишем, как собранные данные покадровой обработки изображений можно оценить с помощью с открытым исходным кодом и свободно доступного программного обеспечения для анализа изображений, Фиджи (или ImageJ), статистически проанализированы с помощью Microsoft Excel и представлены с использованием программного обеспечения GraphPad Prism 14,18. После описания нашего метода, аналогичные эксперименты могут быть разработаны с использованием модифицированных параметров и факторов, чтобы исследовать их влияние на фагосомы созревания; практически любой OTее тип прикрепленных первичных или клеточных линий клеток фагоцитов, генетическая потеря экспериментов функциональных включая ген нокаут и нокдаун, мутанты или выражение слитых белков, фагоцитарных-мишеней, соединений Microbead покрытия, эндосомный / лизосомальных зондов и факторов такого цитокины, химические ингибиторы или миРНК среди других все переменные, которые могут быть применены к основной подход, предусмотренный этим методом. Определим цели и основные требования этого метода следующим образом: <ul type="disc"><li> Цель метода: для прямого, количественный и качественный наблюдение и анализ фагосомы созревания с высоким временным разрешением в живых фагоцитарные клетки. </li><li> Фагоцитарная грузов: надлежащим покрытием микрочастиц или биологической мишенью. Здесь мы используем IgG покрытием микрочастиц сферической полистирола 3 мкм, которые легко усвоенные через Fc-γ рецепторов опосредованного фагоцитоза. Кроме того, любой микроорганизм или микрофона с покрытиемroparticle для которых фагоцитарная рецептор присутствует в клетках могут быть использованы. </li><li> Фагоциты: Приверженец фагоциты, выражающие соответствующие фагоцитарные рецепторы. Здесь мы используем мыши первичные BMMs которые обильно выразить FC-γ рецепторы. В качестве альтернативы, можно использовать дендритные клетки (DC), моноциты или фагоцитирующих эпителиальные клетки или их генетические мутанты или нокаут-варианта. </li><li> Лизосомные грузов: флуоресцентно меченных лизосомные зонд. Здесь, Texas Red-конъюгированные 70000 кДа декстран (Dex70kD) используется в качестве просвета грузом лизосомах. Кроме того, любой флуоресцентно детектируемый маркер клеточной органеллы, или отсека, или соответствующего зонда для phagosomal свойств, таких как кислотность или деструктивного мощности, может быть использован для изучения прогрессирования фагосомы созревания. </li><li> Влияние факторов: Например, сигнальных молекул, химических соединений, генной нокдаун или временную экспрессию мутантных белков может. Здесь мы Forgодин использование такого фактора для простоты, но для недавнего примера с мутантной экспрессии белка и бросовым влияющих факторов размещается на Kasmapour др.. 18 Любой фактор, который может повлиять на фагоцитоз или обстоятельства и мобильность фагосом и / или отсеки, которые взаимодействуют с погашением фагосом потенциально могут быть использованы. </li></ul>

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	<tbody>
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			<td>Dulbecco&#39;s Modified Eagles Medium (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium without phenol red (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>PAA, Austria</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>PAA, Austria</td>
			<td>M11-004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA, Austria</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAA, Austria</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>WillCo-dish&#174; Glass bottom dish (35 mm &#8709;, #1.5)</td>
			<td>WillCo Wells, Netherlands</td>
			<td>GWSt-3522</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLviewTM glass bottom dish, 35 mm</td>
			<td>Greiner Bio one, Germany</td>
			<td>627871</td>
			<td>Alternative: Available as segmented</td>
		</tr>
		<tr>
			<td>Carboxylated latex microspheres</td>
			<td>Polysciences, USA</td>
			<td>#09850</td>
			<td>Available in different diameters, we used 3 &#956;m</td>
		</tr>
		<tr>
			<td>Polystyrene microparticles</td>
			<td>Kisker Biotech, Germany</td>
			<td>PPs-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>ROTH, Germany</td>
			<td>305.1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse whole IgG</td>
			<td>Rockland, USA</td>
			<td>010-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>EDAC crosslinker</td>
			<td>Sigma-Aldrich, USA</td>
			<td>E6383-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris</td>
			<td>Sigma-Aldrich,USA</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ml syringe Omnifix -F</td>
			<td>B.Braun, Germany</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>26&#160;G needle (0.45*25 mm)</td>
			<td>B.Baun, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA, Austria</td>
			<td>E15-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco, USA</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich, USA</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M syringe mounted filter</td>
			<td>Sartorius, Germany</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of phagolysosome biogenesis in live macrophages

Фагоциты играют важную роль в системе врожденного иммунитета путем удаления и ликвидации вторжения микроорганизмов в их фагосом. Фагосомы созревание является сложным и жестко регулируется процесс, при котором зарождается Фагосомы претерпевает радикальные преобразования с помощью хорошо организованных взаимодействий с различных клеточных органелл и отсеков в цитоплазме. Этот процесс, который имеет важное значение для физиологической функции фагоцитирующих клеток по наделяя фагосом с их литических и бактерицидными свойствами, достигает кульминации в слиянии фагосом с лизосом и биогенеза фаголизосомах который считается последним и критический этап созревания для фагосом. В этом докладе мы описываем метод, основанный изображений живых клеток для качественного и количественного анализа динамического процесса лизосом в фагосомы Content Delivery, который является отличительной чертой фаголизосомы биогенеза. Этот подход использует микрошарики IgG-покрытые качестве модели для phagocytosis и флуорофорные-сопряженных декстрана молекулы как просвета лизосом грузов зонда, для того, чтобы следить за динамический доставки контента lysosmal к фагосом в режиме реального времени в живых макрофагов с помощью покадровой обработки изображений и конфокальной лазерной сканирующей микроскопии. Здесь мы подробно описать фон, подготовительные шаги и шаг за шагом экспериментальную установку для включения легко и точно развертывание этого метода в других лабораториях. Наша Описанный метод является простой, надежный, а главное, может быть легко адаптирована для изучения phagosomal взаимодействия и созревание в разных системах и при различных экспериментальных условиях, таких как использование различных типов фагоциты, с потерей функции экспериментов, различных датчиков, а фагоцитарные частиц.

Правильная подготовка клеток и бусы для визуализации имеет решающее значение. Рисунок 1 показывает схему посева, зонда-погрузки и первых шагов изображений в этом методе, основанном на нашем оптимизированного протокола для BMMs. Поэтому крайне важно, что параметры протокола быт?...

Watch this Scientific Journal Video about Study of Phagolysosome Biogenesis in Live Macrophages at JoVE.com

Study of Phagolysosome Biogenesis in Live Macrophages

Изучение фаголизосомы биогенеза в живых макрофагов

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

study-of-phagolysosome-biogenesis-in-live-macrophages

Research

JoVE Journal

Immunology and Infection

14.3K Views.  Helmholtz Centre for Infection Research. Изучение фаголизосомы биогенеза в живых макрофагов

Video: Study of Phagolysosome Biogenesis in Live Macrophages

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Использование оптической ловушки для изучения хост-Возбудитель Взаимодействие для динамических изображений Живая сотовый

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Истощение и разведения макрофагов у мышей

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Создание<em&gt; В пробирке</em&gt; Система по изучению внутриклеточных Поведение<em&gt; Candida glabrata</em&gt; В человека ТНР-1 макрофагов

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Использование<em&gt; Shigella flexneri</em&gt; Для изучения аутофагии-Цитоскелет взаимодействий

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Протеомный профилирования макрофагов 2D электрофореза

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Количественное Цитозольные против вакуолярной<em&gt; Сальмонелла</em&gt; В первичных макрофагах дифференциальной проницаемости

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Фагосоме Миграция и скорость, измеренная в живых первичных макрофагах человека, инфицированных ВИЧ-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Изоляция Salmonella typhimurium-содержащих Phagosomes от макрофагов

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Изоляции и в пробирке культуры мышиных и человека альвеолярные макрофаги

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&amp;#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

Измерение расхода гранулярных производства рос в макрофагах в ответ на FcγR сшивки

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...