<p class="jove_content"&gt; Diese Arbeit wurde vom NIH P20RR018766 und einen Zuschuss von der ABMRF / Foundation for Alcohol Research gefördert.</p

<ol>
	<li>Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+system:+a+vital+link+between+metabolic+syndrome+and+inflammation.">Lymphatic system: a vital link between metabolic syndrome and inflammation.</a> Ann. N. Y. Acad. Sci. 1207, 94-94 (2010).</li><li>Zawieja, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+biology+and+the+microcirculation:+past,+present+and+future.">Lymphatic biology and the microcirculation: past, present and future.</a> Microcirculation. 12, 141-141 (2005).</li><li>Benoit, J. N., Zawieja, D. C., Goodman, A. H., Granger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+mesenteric+lymphatic+pump+and+its+responsiveness+to+acute+edemagenic+stress.">Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.</a> Am. J. Physiol. 257, H2059-H2059 (1989).</li><li>Davis, M. J., Davis, A. M., Ku, C. W., Gashev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+constriction+and+dilation+of+isolated+lymphatic+vessels.">Myogenic constriction and dilation of isolated lymphatic vessels.</a> Am. J. Physiol. Heart. Circ. Physiol. 296, H293-H293 (2009).</li><li>Dougherty, P. J., Davis, M. J., Zawieja, D. C., Muthuchamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensitivity+and+cooperativity+of+permeabilized+rat+mesenteric+lymphatics.">Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1524-R1524 (2008).</li><li>Fay, F. S., Shlevin, H. H., Granger, W. C., Taylor, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin+luminescence+during+activation+of+single+isolated+smooth+muscle+cells.">Aequorin luminescence during activation of single isolated smooth muscle cells.</a> Nature. 280, 506-506 (1979).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+myosin+light+chain+phosphorylation+decreases+rat+mesenteric+lymphatic+contractile+activity.">Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, 726-726 (2009).</li><li>Karaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements,+distribution,+and+functions+in+smooth+muscle.">Calcium movements, distribution, and functions in smooth muscle.</a> Pharmacol. Rev. 49, 157-157 (1997).</li><li>Ratz, P. H., Berg, K. M., Urban, N. H., Miner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+smooth+muscle+calcium+sensitivity:+KCl+as+a+calcium-sensitizing+stimulus.">Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.</a> Am. J. Physiol. Cell. Physiol. 288, C769-C769 (2005).</li><li>Somlyo, A. P., Somlyo, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sensitivity+of+smooth+muscle+and+nonmuscle+myosin+II:+modulated+by+G+proteins,+kinases,+and+myosin+phosphatase.">Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.</a> Physiol. Rev. 83, 1325-1325 (2003).</li><li>Souza-Smith, F. M., Kurtz, K. M., Molina, P. E., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mesenteric+collecting+lymphatic+pump+function+following+acute+alcohol+intoxication.">Adaptation of mesenteric collecting lymphatic pump function following acute alcohol intoxication.</a> Microcirculation. 17, 514-514 (2010).</li><li>Breslin, J. W., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+alters+barrier+function+of+cultured+lymphatic+endothelial+cells+through+a+VEGFR-3-dependent+mechanism.">VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.</a> Lymphat. Res. Biol. 5, 105-105 (2007).</li><li>Shirasawa, Y., Benoit, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-induced+calcium+sensitization+of+rat+lymphatic+smooth+muscle.">Stretch-induced calcium sensitization of rat lymphatic smooth muscle.</a> Am. J. Physiol. Heart. Circ. Physiol. 285, H2573-H2573 (2003).</li><li>Imtiaz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaking+through+Ca2++stores+interacting+as+coupled+oscillators+via+membrane+depolarization.">Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.</a> Biophys. J. 92, 3843-3843 (2007).</li><li>Muller, J. M., Davis, M. J., Kuo, L., Chilian, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+coronary+endothelial+cell+Ca2++concentration+during+shear+stress-+and+agonist-induced+vasodilation.">Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.</a> Am. J. Physiol. 276, 1706-1706 (1999).</li><li>Ferrusi, I., Zhao, J., van Helden, D., von der Weid, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopiazonic+acid+decreases+spontaneous+transient+depolarizations+in+guinea+pig+mesenteric+lymphatic+vessels+in+endothelium-dependent+and+-independent.">Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent.</a> 286, H2287-H2287 (2004).</li></ol>

Keine Interessenskonflikte erklärt.

Eine neuartige Kombination von Methoden wurde eingesetzt, um innere Pumpen von Lymphgefäßen zu studieren. Die Fähigkeit, gleichzeitig zu messen Veränderungen in [Ca 2 +] i und Durchmesser in Pumpen Lymphgefäße wird für die Untersuchung der relativen Beiträge der Ca 2 +-abhängige und Ca 2 +-sensibilisierende Signalwege in der gesamten Mechanismus für die lymphatischen kontraktilen Zyklus. Zulassen Das lymphatische kontraktilen Zyklus beste...

<table border="1"><tbody><tr><td> 1. Ringer 5x Lager </td></tr><tr><td Chemikalie </td><td> Firma </td><td> Katalog-Nummer </td><td> Höhe </td></tr><tr><td> Kochsalz </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Kaliumchlorid </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Calcium Chloride </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Magnesium Sulfate </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> Steriles gefiltertes Wasser </td><td> N / A </td><td> N / A </td><td> Bring to 1 L </td></tr><tr><td> Sterile Filter in autoklaviert Flaschen und speichert bei 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. MOPS-Puffer </td><td></td><td></td><td></td></tr><tr><td> Chemikalie </td><td> Firma </td&gt; <td> Katalog-Nummer </td><td> Höhe </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> Steriles gefiltertes Wasser </td><td> N / A </td><td> N / A </td><td> Bring to 1 L </td></tr><tr><td> Sterile Filter in autoklaviert Flaschen und speichert bei 4 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. Albumin physiologischer Kochsalzlösung (APSS) </td></tr><tr><td> Chemikalie </td><td> Firma </td><td> Katalog-Nummer </td><td> Höhe </td></tr><tr><td> Ringer Lager (5x) </td><td> N / A </td><td> N / A </td><td> 200 ml </td></tr><tr><td> MOPS-Puffer </td><td> N / A </td><td> N / A </td><td> 5 mL </td></tr><tr><td> Sodium Phosphate </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> Natriumpyruvat </td><td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> EDTA-Natrium salt </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Glucose </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> Albumin, Bovine </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> Steriles gefiltertes Wasser </td><td> N / A </td><td> N / A </td><td> Bring to 1 L </td></tr><tr><td> Der pH-Wert auf 7,4 bei 37 ° C, dann Sterilfilter in autoklaviert Flaschen und lagern bei 4 ° C. </td></tr></tbody></table> Tabelle 1. Spezifische verwendeten Reagenzien. Speichern Sie alle bei 4 ° C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

Lymphgefäße aus einem multifunktionalen Transport-System, das Flüssigkeitshomöostase unterhält, liefert Lipide zu den zentralen Kreislauf und wirkt wie ein Überwachungssystem für potenziell schädliche Antigene, die Optimierung der Schleimhaut-Immunität und adaptive Immunantworten 1. Lymphe ist in der interstitiellen Flüssigkeit, die blind endenden initialen Lymphgefäße eintritt, und dann gegen einen Druckgradienten in größeren Sammlung Lymphgefäße transportiert gebildet. Jeder sammelt lymphatischen besteht aus einer Reihe von Segmenten bezeichnet Lymphangiome gemacht, getrennt durch bicuspid Ventile Rückfluss zu verhindern. Jeder Lymphangione besitzt einen kontraktilen Zyklus, der Lymphe gegen ein Druckgefälle in Richtung der zentralen Kreislauf 2 antreibt. Diese phasischen kontraktilen Muster ist analog zu den Herzzyklus mit systolischen und diastolischen Phasen, und mit einem geringeren Rückgang Frequenz 4. Darüber hinaus generiert lymphatische Tonus der glatten Muskulatur und zeigt myogene Verengung und Erweiterung in Reaktion auf Zu-und Abnahmen in luminalen Druck bzw. 5. Ein Hybrid aus molekularen Mechanismen, die sowohl die phasischen und tonischen Kontraktilität Lymphgefäße Unterstützung sind daher vorgeschlagen. Kontraktion der glatten Muskulatur in der Regel durch die cytosolische Ca 2 +-Konzentration reguliert ([Ca 2 +] i) plus Sensibilität für Ca 2 +, der kontraktilen Elemente in Reaktion auf Veränderungen in der Umgebung der Zelle 6. [Ca 2 +] i wird durch die Kombination der Bewegung der Ca 2 + durch Plasmamembran Liganden oder spannungsgesteuerten Ca 2 +-Kanäle und die Freisetzung und Aufnahme von Ca 2 + aus dem internen Speicher bestimmt. Cytosolischen Ca 2 + bindet an Calmodulin und aktiviert Enzyme wie Myosin Light Chain (MLC)-Kinase (MLCK), die wiederum phosphoryliert MLC führt zu Aktin-Myosin-vermittelte Kontraktion 8. Allerdings ist die Sensitivität dieses Weges zu Ca <sup&gt; 2 + kann durch die MLC-Phosphatase (MLKP) 9 geregelt werden. MLCP Aktivität wird durch Rho-Kinase (ROCK) und der Myosin-Phosphatase-Inhibitors Protein CPI-17 geregelt. Hier stellen wir eine Methode, um Veränderungen in auszuwerten [Ca 2 +] i im Laufe der Zeit in isolierten, perfundierten Lymphbahnen um Ca 2 +-abhängige und Ca 2 +-sensibilisierende Mechanismen des lymphatischen Kontraktion der glatten Muskulatur zu studieren. Die Verwendung von isolierten Ratten mesenterialen sammeln Lymphbahnen wir untersucht stretch-induzierten Veränderungen in [Ca 2 +] i und kontraktile Aktivität. Die isolierten Lymph-Modell bietet den Vorteil, dass Druck, Durchfluss, und die chemische Zusammensetzung der Badlösung eng kontrolliert werden kann. [Ca 2 +] i wurde durch das Laden Lymphgefäße mit dem ratiometrisch, Ca 2 +-bindenden Farbstoff Fura-2 bestimmt. Diese Studien werden einen neuen Ansatz für die breitere Problem der Untersuchung der verschiedenen molekularen Mechanismen, die phasischen regulieren bietenKontraktionen im Vergleich Tonic Verengung in lymphatischen glatten Muskulatur.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Wir stellen eine Annäherung an die cytosolische Ca bewerten<sup&gt; 2 +</sup&gt; Konzentration in isolierten Lymphgefäße Ca-Studie<sup&gt; 2 +</sup&gt;-Abhängigen und Ca<sup&gt; 2 +</sup&gt; Sensibilisierend Mechanismen des lymphatischen Kontraktion der glatten Muskulatur.

Die Messung der cytosolischen Ca 2 + In Isolated Kontraktile Lymphgefäße

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

measurement-of-cytosolic-ca2-in-isolated-contractile-lymphatics

Research

JoVE Journal

Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

13.5K Aufrufe.  Louisiana State University Health Sciences Center. Wir stellen eine Annäherung an die cytosolische Ca bewerten 2 + Konzentration in isolierten Lymphgefäße Ca-Studie 2 +-Abhängigen und Ca 2 + Sensibilisierend Mechanismen des lymphatischen Kontraktion der glatten Muskulatur.

Serie: Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Die Messung der γHV68 Infektion bei Mäusen

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Forschung

Immunologie und Infektion

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Die Messung der Antikörper Auswirkungen auf zelluläre Funktion von isolierten Kardiomyozyten

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Messung der taktile Allodynie in einem Mausmodell der bakteriellen Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

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

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

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

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

Die Quantifizierung der Cytosolic vs. Vacuolar<em&gt; Salmonellen</em&gt; In Primäre Makrophagen durch Differential Permeabilisierung

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

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

Messung von<em&gt; In Vitro</em&gt; Integration Aktivität von HIV-1 Preintegration Komplexe

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters: Messung des Durchmessers der Kapsel in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2 + Antworten Imaging während der Shigella Infektion von Epithelzellen

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Messung der Chitinase-Aktivität in biologischen Proben

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

Echtzeitanalyse von Calcium und Kontraktilität in isolierten Lymphgefäßen

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Autor im Rampenlicht: Innovative Methoden in der Lymphödem- und Hypertonieforschung

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

Echtzeit-Auswertung der absoluten, zytosolischen, freien ca2+ und der entsprechenden Kontraktilität in isolierten, druckbeaufschlagten Lymphgefäßen

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...