ברצוננו להודות מתיאס ס דיק, רולנד פ Dreier וסבסטיאן רול לדיון. עבודה זו נתמכה על ידי קתדרת PP00P3_139120 SNSF / 1 וID2153162 פרויקט מענק אוניברסיטת באזל לPB

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We have nothing to disclose.

permeabilization ההפרש הוא שיטה קלה וחזקה כדי לנתח ולכמת את הפצת subcellular של חיידקים פתוגנים בין תאי vacuolar וcytosol. אותו assay שמש בהצלחה עם חיידקים כגון Francisella novicida 2,4 וShigella flexneri 12. עם זאת, מאחר שרבי הפתוגן תאיים לשנות או לשנות את מבני endomembrane מארח, על חוסנו של permeabi...

חיידקים פתוגנים תאיים לשכפל באופן ישיר בcytosol התא המארח, או בתאי vacuolar מיוחדים 1. בשלב הראשוני של הזיהום ביותר פתוגנים לקבל הפנימו או על ידי phagocytosis על ידי תאים מיוחדים (כגון מקרופאגים) או על ידי פעיל בקידום הספיגה שלהם לתאים שאינם phagocytic. בתאי phagocytic, phagosome בדרך כלל משלב עם lysosomes כדי ליצור תא נגרע, שבו חלקיקי phagocytosed מתעכלים. חיידקי cytosolic מיוחדים, כגון Francisella novicida או Shigella flexneri, לברוח השפלה phagosomal ידי גרימת הקרע של phagosome ולאחר מכן לברוח ל2,3 cytosol. זה דורש מנגנון הקשורים ארסי כגון פתוגניות אי Francisella (FPI) או Shigella flexneri T3SS, שמזריק חלבוני מפעיל המקדמים קרע vacuolar 2-4. תכונות cytosol התא המארחמספר הקולטנים זיהוי תבניות נשמרים, כי בדרך כלל לזהות את הנוכחות של פתוגנים ולגרום לחיסון מולדים איתות 5. בנוסף, xenophagy, צורה אנטי-מיקרוביאלי של autophagy יכולה לבזות חיידקים שנכנסים cytosol. חיידקי cytosolic בדרך כלל גם מותאמים ללהקהות או לעקוף תגובות אלה באמצעות אסטרטגיות שונות. לדוגמא, Francisella משנה LPS כדי להימנע מהכרת המארח וShigella מונע גיוס autophagy באמצעות חלבונים מופרשים מפעיל 6,7. אסטרטגיה נוספת לברוח השפלה lysosomal מועסקת על ידי Typhimurium serovar enterica הפתוגן vacuolar המודל סלמונלה, התייחס לאחר מכן כלtyphimurium סלמונלה. סלמונלה משתמשת T3SS להזריק effectors שלשפץ phagosome הראשוני לנישה תאית שלה, סלמונלה המכיל vacuole (SCV) 8,9. מניפולציה מתמשכת של מסלולי מארח היאנחוץ כדי לשמור על vacuole זאת על ידי גיוס שומנים וחומרים מזינים אחרים לvacuole. ואכן, מוטציה SIFA של סלמונלה לא מצליחה לשמור על יציבות vacuolar, ונכנסה cytosol של תאי מארח בתוך שעות לאחר הדבקה, וכתוצאה מכך ההפעלה של מסלולי חיסון מולדים וגיוס autophagy 8. בריחת vacuolar של סלמונלה משתנה בהתאם לסוג התא שהוא לימודים, וכמה מחקרים הראו כי בתאי אפיתל אפילו WT סלמונלה יכולה לברוח SCV וhyperreplicate בcytosol 10. הדיווחים אחרונים מראים כי זיהוי חיסון מולדים של פתוגנים vacuolar גם תלוי ביכולת של המארח להכיר ולערער vacuoles המכיל הפתוגן (PCVs) 11-14. בהתחשב בכך ששניהם המארח או חיידקי גנוטיפ יכול להשפיע על ההפצה של חיידקים תאיים בין vacuolar ותא cytosolic, יש צורך לכמת את מספר לעומת vacuolarחיידקי cytosolic. מאז, בהגדרות ניסיוניות רוב תאי מארח נגועים בחיידק אחד או יותר, ולאחר מכן יכולים נמל כמה חיידקים בתאי subcellular שונים, יש צורך בטכניקה המאפשרת כימות ברמה של חיידקים בודדים. permeabilization ההפרש (הידוע גם בassay הגנת phagosomal) מספק רזולוציה זו 2,4,10,12. Assay מבוסס על permeabilization סלקטיבית של קרום הפלזמה התא המארח עם digitonin חומר הניקוי, מה שמשאיר את קרומי vacuolar שלמים ובכך מאפשר צביעה סלקטיבית של חיידקי cytosolic עם נוגדנים. כאן אנו מספקים שני פרוטוקולים לכימות של cytosolic וvacuolar typhimurium סלמונלה באמצעות permeabilization ההפרש. העיקרון של שיטה זו מתואר באיור 1: מקרופאגים מח העצם נגזרים (BMDMs) נגועים בשלב נייח mCherry להביע סלמונלה. השלב נייח סלמונלה צריכה to לשמש כי הם downregulate הביטוי של T3SS SPI-1, שפעילותם אמורה היה להיות מוכרת על ידי inflammasome NLRC4 ולגרום למוות מהיר של תאים (pyroptosis) של 15 BMDMs. בעקבות זיהום תאים נשטפים וטופלו עם 50 / מיליליטר מיקרוגרם של digitonin דקה 1. תאים נשטפים שוב מייד וטופחו עם נוגדנים נגד סלמונלה מצמידים את FITC לסמן חיידקים עם גישה לcytosol. לאחר שטיפה נוספת תאי צעד הם lysed ואחוז mCherry + (vacuolar) וFITC + / mCherry + (cytosolic) החיידקים נקבע על ידי FACS. כמו כן, אנו מדווחים על הסתגלות של שיטה זו, שניתן להשתמש אם אין זנים להביע חלבון פלואורסצנטי זמינים (איור 2). צעדים נוספים שהוכנסו לאחר תיוג FITC שבו תאים הם קבועים וpermeabilized לחלוטין. לאחר מכן כל חיידקי מגואלות נוגדנים נגד סלמונלה ונוגדנים משני המתאימים. Detשיקוף מכן על ידי מיקרוסקופ במקום ניתוח 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

חיידקים פתוגנים תאיים יכול לשכפל בcytosol או בvacuoles מיוחד המכיל הפתוגן (PCVs). כדי להגיע cytosol, חיידקים כמו Shigella flexneri וFrancisella novicida צריכים לגרום לקרע של phagosome. לעומת זאת, typhimurium סלמונלה משכפל בתא vacuolar, הידוע בשם vacuole סלמונלה המכיל (SCV). מוטציות עם זאת מסוימות של סלמונלה להיכשל כדי לשמור על שלמות SCV ומשתחררים כך לתוך cytosol. אחוז cytosolic לעומת חיידקי vacuolar על הרמה של חיידקים בודדים ניתן למדוד על ידי permeabilization ההפרש, הידוע גם בassay phagosome-ההגנה. הגישה עושה שימוש ברכוש של digitonin חומר הניקוי כדי לאגד כולסטרול באופן סלקטיבי. מאז קרום הפלזמה מכיל יותר כולסטרול מאשר קרומים תאיים אחרים, digitonin יכול לשמש כדי permeabilize סלקטיבי קרום הפלזמה תוך השארת קרומים תאייםבשלמותה. בזיהום קצר, הבא עם הפתוגן לבטא חלבון סמן פלואורסצנטי (למשל mCherry בין השאר), קרום הפלזמה של תאי מארח permeabilized עם דגירה קצרה בdigitonin מכילה מאגר. לאחר מכן תאים נשטפים וטופחו עם נוגדן ראשוני (מצמידים את fluorophore בחירה) המכוון נגד החיידק של בחירה (למשל אנטי סלמונלה -FITC) ושטפו שוב. אם חיידקים לא מסומנים משמשים, ניתן לעשות צעד נוסף, שבו כולם קרומי permeabilized וכל החיידקים מוכתמים עם נוגדן מקביל. בעקבות הצביעה, אחוז חיידקי vacuolar וcytosolic ניתן לכמת על ידי FACS או במיקרוסקופ על ידי ספירת אירועים חד או דו-חיוביים. כאן אנו מספקים פרטים ניסיוניים לשימוש בטכניקה זו עם typhimurium סלמונלה החיידק. היתרון של assay זה הוא כי, בניגוד לassay האחר, היא מספקת כימות ברמה של bacte אחתריה, ואם נותח על ידי מיקרוסקופ מספק את המספר המדויק של cytosolic וחיידקים vacuolar בתא נתון.

איור 1 ואיור 2 מציגים את השרטוטים של assay digitonin המתוארים בפרוטוקול 1 ופרוטוקול 2 ממחישים את השלבים הקריטיים בפרוטוקול ותוצאות שהתקבלו. איור 3 מראה תוצאות FACS טיפוסיות. בקרות חיוביות ושליליות משמשות כדי להגדיר את השערים לחיידקים mCherry + / FITC- (vacuolar סלמונלה) וmCherry + /...

Watch this Scientific Journal Video about Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization at JoVE.com

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.

כימות של Cytosolic לעומת vacuolar<em&gt; סלמונלה</em&gt; ביסודי המקרופאגים על ידי דיפרנציאל permeabilization

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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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.

מדידה של Cytosolic Ca 2 + ב lymphatics כויץ מבודד

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.

מידות תא יחיד של קרע vacuolar נגרמים על ידי פתוגנים תאיים

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.

Assay תפוקה גבוהה להפנוטיפ<em&gt; Enterica סלמונלה</em&gt; Typhimurium איגוד, פלישה, ושכפול בהמקרופאגים

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.

Transfection יעיל ביותר של של אדם המקרופאגים THP-1 על ידי 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.

Proteomic פרופיל של המקרופאגים על ידי 2D אלקטרופורזה

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.

הגירה ו מהירות Phagosome נמדד המקרופאגים אדם מן המעלה הראשונה חי עם 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 ...

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.

אפיון פנוטיפי של מקרופאגים כליה עכברוש ידי cytometry זרימה

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.

בידוד של סלמונלה typhimurium-המכיל Phagosomes של מקרופאגים

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.

זמן לשגות הדמיה תלת-ממדית של Phagocytosis על ידי העכבר מקרופאגים

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.

כימות של נוגדנים תלויי שיפור של הנגיף Zika בתאי אדם ראשי

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

כימות של Cytosolic לעומת vacuolar

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מדידה של Cytosolic Ca^{2 +} ב lymphatics כויץ מבודד