Мы хотели бы поблагодарить Mathias S. Дик, Roland Ф. Дрейер и Себастьян Рул для обсуждения. Эта работа была поддержана SNSF профессуру PP00P3_139120 / 1 и Университета Базеля грантового проекта ID2153162 к ПБ

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We have nothing to disclose.

Дифференциальный пермеабилизации это легко и надежный метод для анализа и количественной оценки субклеточную распределение бактериальных патогенов между вакуолярных отсеков и цитозоле. Анализ же успешно используется такими бактериями, как Francisella novicida 2,4 и шигеллы Флекс...

Внутриклеточные бактериальные патогены репликации либо непосредственно в цитозоль клетки-хозяина, или в специализированных вакуолярных отсеков 1. Во время начальной стадии инфекции большинство патогенных микроорганизмов получают усвоены либо фагоцитоза специализированных клеток (например, макрофагов), либо активно продвигает свою собственную поглощение в не-фагоцитирующих клеток. В фагоцитирующих клеток, как правило, фагосомы сливается с лизосомы сформировать деструкции отсек, где фагоцитированы частицы переваренной. Специализированный цитозольные бактерии, такие как Francisella novicida или Shigella Флекснера, бежать деградации phagosomal, вызывая разрыв фагосомы и впоследствии бежать в цитозоле 2,3. Это требует вирулентности-ассоциированных механизм таких как Francisella острова патогенности (FPI) или Shigella Флекснера T3SS, который впрыскивает эффекторные белки, которые способствуют вакуолярной разрыв 2-4. Особенности клетка-хозяин цитозольныеКоличество сохраняемых распознающих рецепторов, которые обычно признают наличие возбудителей и вызывают иммунной сигнализации 5. Кроме того, xenophagy, антимикробное форма аутофагии может ухудшить бактерии, которые попадают в цитозоль. Цитозольные бактерии, как правило, хорошо приспособлены к притупить или обойти эти ответы с помощью различных стратегий. Например, Francisella изменяет свои LPS, чтобы избежать признания хоста и шигеллы предотвращает набор аутофагии с помощью секретируемых белков эффекторные 6,7. Другая стратегия, чтобы избежать деградации лизосомами используется в модели вакуолярный возбудителя Salmonella enterica серовар Typhimurium, после называют Salmonella Typhimurium. Сальмонелла использует T3SS чтобы придать эффекторы, что переделывать первоначальный фагосомы в его внутриклеточной ниши, сальмонеллы -содержащего вакуоли (ПЗК) 8,9. Непрерывная манипуляции принимающих путей являетсянеобходимо сохранить эту вакуоль по вербовке липидов и других питательных веществ в вакуоли. Действительно, SIFA мутант Salmonella не удается поддерживать стабильность вакуолярной, и входит в цитозоле клеток-хозяев в течение нескольких часов после заражения, что приводит к активации врожденного иммунитета путей и аутофагии набора 8. Вакуолярной побег Salmonella варьируется в зависимости от типа клеток, что является исследования, и несколько отчетов показали, что в эпителиальных клетках даже БТ Сальмонелла может избежать SCV и hyperreplicate в цитозоле 10. Последние отчеты показывают, что врожденная иммунная обнаружения патогенов вакуолярных также зависит от способности хозяина, чтобы признать и дестабилизировать патогенов, содержащий вакуоли (PCVs) 11-14. Учитывая, что оба хоста или бактерии генотип может влиять на распределение внутриклеточных бактерий между вакуолярную и цитозольной отсека, необходимо определить число вакуоли VS.цитозольные бактерии. Так, в большинстве экспериментальных установок клетки-хозяева инфицированы более чем одной бактерии, и в дальнейшем может питать несколько бактерий в различных субклеточных отсеков, метод необходим, что позволяет количественной на уровне отдельных бактерий. Дифференциальный пермеабилизации (также известный как защита phagosomal анализа) обеспечивает эту резолюцию 2,4,10,12. Анализ основан на селективной проницаемости мембраны клетки-хозяина плазмы с моющим средством дигитонин, что оставляет нетронутыми вакуоли мембраны и, таким образом, позволяет избирательно окрашивание цитоплазмы бактерии с антителами. Здесь мы предлагаем два протокола для количественного определения цитозольного и вакуолярной Salmonella Typhimurium, используя дифференциальный пермеабилизации. Принцип этого метода описан в рисунке 1: костный мозг, полученные макрофаги (BMDMs) заражены стационарной фазы mCherry-выражения сальмонеллы. Стационарная фаза сальмонеллы нужно тО быть использованы, потому что они подавляют экспрессию SPI-1 T3SS, чья деятельность в противном случае будут признаны инфламмасома NLRC4 и вызывают быструю смерть клеток (pyroptosis) в BMDMs 15. После инфекции клетки промывают и обрабатывают 50 мкг / мл дигитонина в течение 1 минуты. Клетки снова промывают сразу и инкубировали с анти-антител сальмонеллы, соединенных с FITC, чтобы отметить бактерии с доступом в цитозоль. После дополнительного этапа промывки клетки лизируют и процент mCherry + (вакуоли) и FITC + / + mCherry (цитозольного) бактерий определяется FACS. Мы также сообщают об адаптации этого метода, которые могут быть использованы, если нет флуоресцентные штаммы, экспрессирующие белок не доступны (рисунок 2). Дополнительные шаги вводятся после FITC маркировки, в котором клетки фиксированной и полностью проницаемыми. После этого все бактерии окрашивали анти Salmonella антител и соответствующих вторичных антител. Detотражения затем сделать с помощью микроскопии вместо анализа FACS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td>50ug/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>mpbio</td>
			<td>219998380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>791733</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1-FITC</td>
			<td>KPL</td>
			<td>01-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1</td>
			<td>KPL</td>
			<td>02-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Calnexin</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-860D</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>anti-PDI</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-890</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody-488</td>
			<td>Molecular Probes</td>
			<td>A-11070</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15710-49</td>
			<td>100ug/mL and 10ug/mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5141</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Amimed</td>
			<td>5-13K00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>imaging done at 63x</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection mCherry 610 nm</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection FITC 530 nm</td>
		</tr>
	</tbody>
</table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

Внутриклеточные бактериальные патогены могут размножаться в цитоплазме или в специализированных патогенов, содержащих вакуолей (PCVs). Чтобы добраться до цитозоль, бактерии, как Shigella Флекснера и Francisella novicida нужно вызвать разрыв фагосомы. В отличие, сальмонелла Typhimurium реплицируется в вакуолярной купе, известный как Salmonella -содержащего вакуоли (SCV). Однако некоторые мутанты Salmonella не поддерживать SCV целостность и, таким образом, выпущен в цитозоль. Процент цитозольного против вакуолярных бактерий на уровне отдельных бактерий может быть измерена дифференциальной проницаемости, также известный как фагосомы-анализ защиты. Подход делает использование имущества моющего средства дигитонина селективно связываться холестерина. Поскольку плазматической мембраны содержит больше холестерина, чем других клеточных мембран, дигитонин может быть использован, чтобы селективно проницаемыми плазматическую мембрану, оставляя внутриклеточных мембранбез изменений. Короче говоря, после инфицирования патогеном, экспрессирующих флуоресцентный белок-маркер (например mCherry среди прочих), плазматической мембраны клеток-хозяев в проницаемыми с короткой инкубации в дигитонин, содержащей буфер. Затем клетки промывали и инкубировали с первичным антителом (соединенный с флуорофора выбора), направленный против бактерии выбора (например, анти-Salmonella -FITC) и снова промывают. Если немаркированных бактерии используется дополнительная стадия может быть сделано, в котором все мембраны проницаемыми и все бактерии окрашивали с соответствующим антителом. После окрашивания, процент вакуолярных и цитоплазмы бактерий может быть количественно FACS или микроскопа путем подсчета одинарные или двойные-положительных событий. Здесь мы предлагаем экспериментальные детали для использования этой техники с бактерией Salmonella Typhimurium. Преимущество такого анализа является то, что, в отличие от других анализа, он обеспечивает количественную на уровне одного bacteРИА, и если анализировали с помощью микроскопии обеспечивает точное число цитозольного и вакуолярных бактерий в данной клетке.

На рисунке 1 и 2 приведены схемы дигитонинового анализа, описанные в протокол 1 и протокол 2, иллюстрирующий важные шаги в протоколе и полученные результаты. Рисунок 3 показывает типичные FACS результаты. Положительные и отрицательные элементы управления используются для ?...

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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 ...

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Research

JoVE Journal

Immunology and Infection

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

10.8K Views.  University of Basel. We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

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

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Измерение цитозольного Са 2 + В изолированном Сократительная лимфатических

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Единичных измерений Сотовые вакуолярных разрыв под действием внутриклеточных патогенов

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Высокая пропускная Анализ фенотипу<em&gt; Salmonella enterica</em&gt; Typhimurium Ассоциация, Нашествие, и репликации в макрофагах

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Высокоэффективная Трансфекцию человека THP-1 макрофагов по Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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 ...

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 ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Фенотипическая Характеристика макрофаги почек крыс с помощью проточной цитометрии

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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. ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Промежуток времени 3D визуализации фагоцитоза макрофагами, мыши

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Количественное определение антител зависимых повышение Зика вируса в первичных клеток человека

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...