The authors would like to thank the following funding sources: RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806, 1R01DA034582 (to S.R) and F31 DA026264-01A1, T32 DA07097 (to J.N.).

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The authors have no competing financial interests.

القيود الرئيسية للتقنية فلوروميتريك هي التباين التجريبية فضلا عن فقدان الخلايا المرتبطة الغسيل واستخدام الخلايا غير ملتصقة واسعة النطاق. ويلاحظ التباين إلى حد كبير بسبب الاختلاف في جزيئات الفلورسنت كما وزنها وpipetting لأثناء إعد?...

الكمي للإشارة الفلورسنت وقد استخدم على نطاق واسع في العديد من الأساليب العلمية التي تتراوح بين PCR، التدفق الخلوي، الفحص المجهري متحد البؤر، والحنق التحليل لمعدد ELISA. التصوير مضان وتقدير له تطبيق واسع النطاق ويمكن أن يكون أداة عظيمة للتحليل الكمي من العمليات الخلوية المختلفة. وقد أحدثت ثورة استخدام علامات الفلورسنت وإشارة في العقد الماضي، وظهور القراء لوحة الفلورسنت يسر الكمي إنتاجية عالية من مضان المنبعثة خلال العمليات الخلوية. تحليل فلوروميتريك يمكن أن تكون بمثابة أداة عظيمة في تقدير حجم البلعمة. وقد تمت دراسة البلعمة منذ اكتشاف البالعات التي كتبها ميتشنيكوف في عام 1800 1. وعلى مر السنين، وقد تم استخدام مجموعة متنوعة من الأساليب لدراسة هذه العملية الهامة ضرورية للدفاع ضد غزو المناعة الفطرية البكتيرية، الفيروسية، الفطرية والطفيلية الممرضة 2-5. الأساليب السابقة من تقدير تستخدم المجهري وتقنيات stereological من أجل تصور الخلايا التي يتم phagocytosing، ثم جرى كميا عن طريق عد من الجسيمات المنضوية (يدويا أو باستخدام البرمجيات) 6-8. بعض العيوب باستخدام المجهر وحده للتحليل الكمي هي أن العد والفرز اليدوي من البكتيريا هو عمالة كثيفة وأكثر عرضة للانحياز مراقب. طريقة أخرى تستخدم في القياس الكمي للالبلعمة هي التقنية الميكروبيولوجية من الطلاء من البكتيريا من الخلية لست] (بعد البلعمة) على لوحات ثقافة البكتيرية، ولكن هذا الأسلوب يمكن أن تفشل لحساب آليات للجراثيم وجود البكتيريا المنضوية جزئيا. هذا الأسلوب هو المزيد من العمل المكثف مقارنة المجهري ويأخذ عدة أيام لتحليلها. التدفق الخلوي يبدو أن يكون أسرع وأنجع وسيلة لقياس البلعمة وتم استخدامها من قبل العديد من الجماعات 9-11، ولكن التكلفة العالية المرتبطة عادة مع فيstrument اللازمة لتحليل يجعل من الأسلوب الأكثر مكلفة بالمقارنة مع المقايسات التي سبق ذكرها. طريقة فلوروميتريك هو بديل جيد لالتدفق الخلوي لتحليل الجسيمات الداخلي لأنه يقدم الكمي غير منحازة من مضان باستخدام المعدات التي ليس كما باهظة التكلفة. فوائد إضافية أخرى من التألق وكفاءة عالية، وقدرات إنتاجية عالية لقياس مضان المنبعثة من جزيئات المنضوية المسمى. فوائد التألق يمكن استقراء لتقدير حجم العمليات الأخرى من البلعمة. على سبيل المثال، تحليل فلوروميتريك يمكن تطبيقها لدراسة أي عملية تؤدي إلى تغييرات في التعبير عن داخل الخلايا أو مستقبلات غشاء محدد، والتغيرات في نفاذية الخلية / الجدوى والفعالية ترنسفكأيشن، وتعديل في الأكتين البلمرة. واحد السلبي لتقنية فلوروميتريك هو أنه، اعتمادا على التسميات المستخدمة، قد يكون هناك ارتفاعتجربة لتجربة تقلب التي يمكن عادة أن تحل من خلال إظهار البيانات من خلال الكمي النسبي، مثل تغير أضعاف أو الزيادة في المئة من السيطرة.

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		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:131px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">1-&#956;m yellow-green fluorescent Fluo-Spheres;</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">F8852</td>
			<td style="width:255px;">combine 50&#956;l with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Heat killed E. coli BioParticles fluorescein conjugate</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2861</td>
			<td style="width:255px;">reconstitute (5 mg) in 50&#956;l of PBS and combine with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Opsonizing reagent</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2870</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rhodamine phalloidin</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">R415</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Trypan blue</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">the items below are available in many brands but the items we used in this study are from the following manufacturers</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">RPMI Cell growth media&#160;</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Supplemented with 10% FBS and 1% pennicillin/Streptomycin; warm in 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fluorescent plate reader-Fluostar Omega</td>
			<td style="width:131px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Paraformaldehyde (16%)</td>
			<td style="width:131px;">Fischer Scientific</td>
			<td>AA433689M</td>
			<td style="width:255px;">dilute to 4% before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">96 well plates</td>
			<td style="width:131px;">Greiner</td>
			<td style="width:119px;">655097</td>
			<td style="width:255px;">clear or black or clear bottom - black plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Multichannel pipette (8-12 channels)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Reagent reservoirs</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1x PBS</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microfuge tubes (0.6 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">&#160;Conicles (10 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

والهدف من التحليل فلوروميتريك هو بمثابة وفعالة من حيث التكلفة، طريقة إنتاجية عالية الكفاءة في تحليل البلعمة والعمليات الخلوية الأخرى. هذه التقنية يمكن استخدامها على مجموعة متنوعة من أنواع الخلايا، سواء ملتصقة وغير ملتصقة، لدراسة مجموعة متنوعة من الخصائص الخلوية. عند دراسة البلعمة، تقنية فلوروميتريك تستخدم أنواع الخلايا البلعمية مثل الضامة، والجزيئات opsonized fluorescently المسمى الذي يمكن أن تسقط في وجود الأزرق التريبان مضان. وبعد الطلاء الضامة ملتصقة في لوحات 96-جيدا، والجسيمات الفلورية (الأخضر أو ​​الأحمر) تدار ويتم السماح خلايا في phagocytose لكميات متنوعة من الوقت. وبعد استيعاب جزيئات الفلورسنت، ويتم غسلها الخلايا مع الأزرق التريبان، مما يسهل انقراض إشارة الفلورسنت من البكتيريا التي لا المنضوية، أو مجرد التمسك سطح الخلية. بعد غسل التريبان، يتم غسلها الخلايا مع PBS، ثابتة، وهيالثانية ملطخة دابي (التسمية الفلورسنت الزرقاء النووية)، والذي يعمل على تسمية نوى الخلايا. من جانب الكمي فلوروميتريك بسيط من خلال لوحة قراءة النووي (الأزرق) أو الجسيمات (أحمر / أخضر) مضان يمكننا فحص نسبة الوحدات مضان النسبية للأخضر: أزرق وتحديد مؤشر أكلة يدل على كمية من البكتيريا الفلورية المنضوية لكل خلية. مدة الفحص باستخدام طريقة والأقنية 96-جيدا الماصات لغسل، من نهاية البلعمة لوضع حد من الحصول على البيانات، هو أقل من 45 دقيقة. التدفق الخلوي يمكن أن تستخدم بطريقة مماثلة ولكن الاستفادة من التألق هو الإنتاجية العالية، طريقة سريعة للتقييم مع التلاعب الحد الأدنى من العينات وتقدير سريع للكثافة الفلورسنت لكل خلية. استراتيجيات مماثلة يمكن تطبيقها على الخلايا غير ملتصقة والبكتيريا وصفت العيش، الأكتين البلمرة، وأساسا لأي عملية الاستفادة من مضان. ولذلك، التألق هي طريقة واعدة لتكلفة منخفضة، throughp أعلى مستوياتهقدرات UT في دراسة العمليات الخلوية.

بطريقتين رئيسيتين من قياس البلعمة والبلمرة أكتين اللاحقة من خلال استخدام هذا البروتوكول هو لمراقبة مستمرة البلعمة أو متزامنة. ويوضح التحليل المجهري (الشكل 1B) صور الفلورسنت مماثلة لما التألق وتسجيل (انظر أيضا الشكل ...

Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

تقنية عالية الإنتاجية فلوروميتريك تقييم بلعم البلعمة وأكتين البلمرة

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

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Immunology and Infection

14.5K Views.  University of Minnesota. Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Video: High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

الإنتاجية العالية لطريقة الكشف عن فيروس الإنفلونزا

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

الإنتاجية العالية للفحص واسع الطيف الكيميائية مثبطات الحمض النووي الريبي الفيروسات

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

بلعم الكولسترول استنفاد وتأثيره على البلعمة من<em&gt; المستخفية المورمة</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

وإنتاجية عالية الفحص المدعم لتقييم فعالية الدواء ضد بلعم Passaged<em&gt; المتفطرة السلية</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

الخلية الحرة البيوكيميائية المقايسة الانزيمية فلوروميتريك لقياس الفائق وبيروكسيد في البروتين الدهني العالي الكثافة

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

استخدام تعزيز الفلورية الخضراء البروتين-الإعراب عن الإشريكيّة القولونية لتقييم البلعمه بلعم البريتوني الماوس

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

إنتاجية عالية في تقييم المختبر من الكمون عكس الوكلاء عن فيروس نقص المناعة البشرية النسخ والربط

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

البلعمه البلاعم السنخية وإزالة البكتيريا في الفئران

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

في تقييم فيفو الضامة السنخيه افيروسيتوسيس بعد التعرض للأوزون

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.