Name: essential components of borreliella (borrelia) burgdorferi in vitro transcription assays
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Dette arbejde blev støttet af Health Sciences Strategic Investment Fund Faculty Development Grant fra Creighton University. B. burgdorferi RpoC-His10X-stammen blev venligst leveret af Dr. D. Scott Samuels fra University of Montana. E. coli-stammen, der huser pMAL-C5X-plasmidet, der koder for en maltosebindende proteinmærket rpoD-allel, blev venligt leveret af Dr. Frank Gherardini fra Rocky Mountain Laboratories, NIAID, NIH.

1. Oprensning af RNA-polymerasen og fremstilling af RNA-polymerasestamme
<ol><li>Cellepellet opsamles fra 2-4 L B. burgdorferi RpoC-His10X dyrket i BSKII-medium i et mikroaerofilt miljø (34 °C, 5% CO 2, 3% O 2) til en densitet på2-4 x 107 celler / ml ved hjælp af tidligere beskrevne celleindsamlingsprotokoller28,29. Der centrifugeres ved 10.000 x g ved 4 °C i 30 minutter i 500 ml centrifug...

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DksA-dependent+regulation+of+RpoS+contributes+to+Borrelia+burgdorferi+tick-borne+transmission+and+mammalian+infectivity.">DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity.</a> PLoS Pathogen. 17 (2), 1009072 (2021).</li><li>Boyle, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+RNA+polymerase+transcription+system:+a+new+tool+to+study+transcriptional+activation+in+Borrelia+burgdorferi.">Establishment of an in vitro RNA polymerase transcription system: a new tool to study transcriptional activation in Borrelia burgdorferi.</a> Scientific Reports. 10 (1), 8246 (2020).</li><li>Silar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+in+vitro+transcription+system+for+analysis+of+Corynebacterium+glutamicum+promoters+recognized+by+two+sigma+factors.">Use of in vitro transcription system for analysis of Corynebacterium glutamicum promoters recognized by two sigma factors.</a> Current Microbiology. 73 (3), 401-408 (2016).</li><li>Maciag, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcription+profiling+of+the+sigmaS+subunit+of+bacterial+RNA+polymerase:+re-definition+of+the+sigmaS+regulon+and+identification+of+sigmaS-specific+promoter+sequence+elements.">In vitro transcription profiling of the sigmaS subunit of bacterial RNA polymerase: re-definition of the sigmaS regulon and identification of sigmaS-specific promoter sequence elements.</a> Nucleic Acids Research. 39 (13), 5338-5355 (2011).</li><li>Chang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRENDA,+the+ELIXIR+core+data+resource+in+2021:+New+developments+and+updates.">BRENDA, the ELIXIR core data resource in 2021: New developments and updates.</a> Nucleic Acids Research. 49, 498-508 (2021).</li><li>Samuels, D. 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In vitro transkriptionsassays konstrueret ved hjælp af den præsenterede protokol blev for nylig brugt til at studere rollen som en transkriptionsfaktor i B. burgdorferi og kan anvendes til at opbygge lignende eksperimenter ved hjælp af andre transkriptionsfaktorer, sigmafaktorer og molekyler23. Når aktiv RNA-polymerase fra B. burgdorferi er opnået og dens aktivitet detekteret, kan komponenter og betingelser inden for in vitro-transkriptionsassays ændres. A...

Borreliose og attakvis feber skyldes spirochetepatogener i slægterne Borrelia og Borreliella 1,2,3. Borreliose er en fremtrædende vektorbåren sygdom i Nordamerika, og derfor er Borreliella burgdorferi en fremtrædende modelorganisme til at studere spirochetebiologi 4,5. Undersøgelser af B. burgdorferi-mekanismerne for transkriptionsregulering sigter mod bedre at forstå dens tilpasninger til ændringer i miljøet, da den cykler mellem sin flåtvektor og pattedyrværter 6,7. Ændringer i pH, temperatur, osmolaritet, næringsstoftilgængelighed, kortkædede fedtsyrer, organiske syrer og opløst ilt og kuldioxidniveauer modulerer ekspressionen af gener, der er vigtige for, at B. burgdorferi kan overleve i sin leddyrvektor og inficere dyr 8,9,10,11,12,13,14,15,16,17,18. At forbinde disse reaktioner på stimuli med reguleringsmekanismer har været et vigtigt aspekt af B. burgdorferi-forskningen 19.
Transskriptionsfaktorer og sigmafaktorer styrer transkriptionen af gener, der udfører cellulære processer. Lyme og recidiverende feber spirochetes har et relativt sparsomt sæt transkriptionsfaktorer og alternative sigma-faktorer. På trods af dette er der komplekse transkriptionelle ændringer, der styrer B. burgdorferis svar på miljøet20,21,22. De specifikke mekanismer, der driver transkriptionelle ændringer i B. burgdorferi som reaktion på miljøændringer, forbliver uklare. In vitro transkriptionsassays er kraftfulde værktøjer til at anvende en biokemisk tilgang til at analysere funktionen og reguleringsmekanismerne for transkriptionsfaktorer og sigmafaktorer23,24,25,26.
Et in vitro transkriptionsassaysystem under anvendelse af B. burgdorferi RNA-polymerase blev for nylig etableret24. Da bakterier ofte har unikke cellulære fysiologier, reagerer RNA-polymeraser af forskellige arter og slægter forskelligt på enzymoprensning, enzymlagring og reaktionsbufferbetingelser27. B. burgdorferi er også genetisk fjernt fra de mange bakteriearter, hvor RNA-polymeraser er blevet undersøgt20. Aspekter af enzympræparation, såsom lysis-, vaske- og elueringsbufferbetingelser, opbevaringsbuffer, in vitro-transkriptionsreaktionsbuffer og metoden til analysekonstruktion kan alle ændre RNA-polymeraseaktivitet. Heri leverer vi en protokol til oprensning af RNA-polymerase og sigmafaktor RpoD, produktion af lineær dobbeltstrenget DNA-skabelon og konstruktion af in vitro-transkriptionsassays for at lette reproducerbarhed mellem laboratorier, der bruger dette system. Vi beskriver detaljeret et eksempel på en reaktion for at demonstrere det lineære område for RpoD-afhængig transkription og diskuterer begrænsninger og alternativer til denne tilgang.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 micron syringe filter</td>
			<td>Thermo Scientific</td>
			<td>726-2545</td>
			<td>Step 1.7 and 2.3</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>MidSci</td>
			<td>C50B</td>
			<td>Step 1.3, and subsequent steps</td>
		</tr>
		<tr>
			<td>50 mL high-speed centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>3119-0050PK</td>
			<td>Step 1.2</td>
		</tr>
		<tr>
			<td>500 mL Centrifuge bottles</td>
			<td>Thermo Scientific</td>
			<td>3120-9500PK</td>
			<td>Step 1.1</td>
		</tr>
		<tr>
			<td>B-PER and instruction manual</td>
			<td>Thermo Scientific</td>
			<td>78248</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>Step 2.6</td>
		</tr>
		<tr>
			<td>Centrifugal filters 10 Kd cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC8010</td>
			<td>Step 1.11 and 2.11</td>
		</tr>
		<tr>
			<td>Cobalt resin and instruction manual</td>
			<td>Thermo Scientific</td>
			<td>89969</td>
			<td>Step 1.9</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Acros Organics</td>
			<td>426380500</td>
			<td>Step 1.4 and subsequent steps</td>
		</tr>
		<tr>
			<td>Dnase (Nuclease)</td>
			<td>Millipore Sigma</td>
			<td>70746</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Factor Xa Protease&#160;</td>
			<td>Haematologic Technologies</td>
			<td>HCXA-0060</td>
			<td>Step 2.6</td>
		</tr>
		<tr>
			<td>GE Typhoon 5 Phosphoimager</td>
			<td>GE lifesciences</td>
			<td>Multiple</td>
			<td>Step 4.15</td>
		</tr>
		<tr>
			<td>Gel Imager</td>
			<td>Bio-Rad</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>H2O for in vitro transcription</td>
			<td>Fisher Scientific</td>
			<td>7732-18-5</td>
			<td>Step 3.2 and 3.3</td>
		</tr>
		<tr>
			<td>high fidelity PCR kit</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td>Step 3.1</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Step 1.1, and subsequent steps</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 5 x 1 mL</td>
			<td>Cytiva Life Sciences</td>
			<td>17040601</td>
			<td>Step 2.8</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>56750-100G</td>
			<td>Step 1.9</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Thermo Scientific</td>
			<td>90082</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Magnesium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>S25401</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Manganese chloride</td>
			<td>Fisher Scientific</td>
			<td>S25418</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Mini protean tetra cell</td>
			<td>Bio-Rad</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Thermo Scientific</td>
			<td>85124</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>NTP mixture</td>
			<td>Thermo Scientific</td>
			<td>R0481</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td>Step 3.2</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>MidSci</td>
			<td>PR-PCR28ACF</td>
			<td>Step 1.12</td>
		</tr>
		<tr>
			<td>PD-10 Sephadex buffer exchange column and instruction manual</td>
			<td>Cytiva</td>
			<td>17085101</td>
			<td>Step 1.10 and 2.10 (gel filtration column)</td>
		</tr>
		<tr>
			<td>pMAL Protein Fusion 
			and Purification System Instruction manual</td>
			<td>New England Biolabs</td>
			<td>E8200S</td>
			<td>Step 2.1</td>
		</tr>
		<tr>
			<td>Polyacrylamide gels AnyKD</td>
			<td>Bio-Rad</td>
			<td>456-8125</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>Potassium glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1251</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Thermo Scientific</td>
			<td>78425</td>
			<td>Step 1.4 and 2.2</td>
		</tr>
		<tr>
			<td>Radiolabeled ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU503H</td>
			<td>Step 4.2</td>
		</tr>
		<tr>
			<td>RNA Loading Dye, (2x)</td>
			<td>New England Biolabs</td>
			<td>B0363S</td>
			<td>Step 4.13</td>
		</tr>
		<tr>
			<td>Rnase inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>Step 4.1</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biotek</td>
			<td>Mutiple</td>
			<td>Step 1.13 and subsequent protien quality check steps</td>
		</tr>
		<tr>
			<td>TBE-Urea gels 10 percent</td>
			<td>Bio-Rad</td>
			<td>4566033</td>
			<td>Step 4.14</td>
		</tr>
	</tbody>
</table>

essential components of borreliella (borrelia) burgdorferi in vitro transcription assays

Borreliella burgdorferi er et bakterielt patogen med begrænsede metaboliske og genomiske repertoirer. B. burgdorferi passerer ekstracellulært mellem hvirveldyr og flåter og ombygger dramatisk sin transkriptionsprofil for at overleve i forskellige miljøer under infektion. Et fokus for B. burgdorferi undersøgelser er klart at forstå, hvordan bakterierne reagerer på sit miljø gennem transkriptionelle ændringer. In vitro transkriptionsassays gør det muligt at dissekere de grundlæggende mekanismer for transkriptionsregulering. Her præsenterer vi en detaljeret protokol, der beskriver B. burgdorferi RNA-polymeraseoprensning og opbevaring, sigmafaktoroprensning, DNA-skabelongenerering og in vitro-transkriptionsassays . Protokollen beskriver brugen af RNA-polymerase oprenset fra B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC er en tidligere offentliggjort stamme, der indeholder et 10XHis-tag på rpoC-genet , der koder for den største underenhed af RNA-polymerasen. In vitro transkriptionsassays består af RNA-polymerasen oprenset fra stamme 5A4-RpoC, en rekombinant version af housekeeping sigmafaktoren RpoD og en PCR-genereret dobbeltstrenget DNA-skabelon. Mens proteinrensningsteknikkerne og tilgangene til samling af in vitro-transkriptionsassays er konceptuelt velforståede og relativt almindelige, er håndteringsovervejelser for RNA-polymeraser ofte forskellige fra organisme til organisme. Protokollen, der præsenteres her, er designet til enzymatiske undersøgelser af B. burgdorferi RNA-polymerasen. Metoden kan tilpasses til at teste transkriptionsfaktorernes, promotorernes og posttranslationelle modifikationers rolle på RNA-polymerasens aktivitet.

I en in vitro transkriptionsreaktion, hvor reaktionens begrænsende trin er sigmafaktormedieret transkriptionsinitiering, bør transkriptionsaktiviteten øges lineært med mængden af sigmafaktor. Vi præsenterer forberedelsen af in vitro-transkriptionseksperimenter, der tester en række RpoD-koncentrationer sammen med to koncentrationer af RNA-polymerase for at observere det resulterende varierende signal fra radioaktivt mærket nukleotidinkorporering i RNA-produkter. Repræsentative resultater af frem...

Watch this Scientific Journal Video about Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays at JoVE.com

Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

In vitro transkriptionsassays kan dechiffrere mekanismerne for transkriptionel regulering i Borreliella burgdorferi. Denne protokol beskriver trinene til oprensning af B. burgdorferi RNA-polymerase og udførelse af in vitro-transkriptionsreaktioner . Eksperimentelle tilgange ved anvendelse af in vitro transkriptionsassays kræver pålidelig oprensning og opbevaring af aktiv RNA-polymerase.

Væsentlige komponenter i Borreliella (Borrelia) burgdorferi In vitro transkriptionsassays

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

In vitro transkriptionsanalysesystemet for Borreliella burgdorferi giver et biokemisk værktøj til efterforskere til at studere genreguleringsmekanismer og faktorer, der styrer den enzymatiske aktivitet af RNA-polymerase. Systemet giver os mulighed for at teste, hvordan transkriptionsfaktorer, co-faktorer, saltkoncentrationer og pH påvirker Borreliella burgdorferi RNA-polymerasefunktionen, hvilket bidrager til vores overordnede forståelse af genregulerende mekanismer. Denne kraftfulde teknik kan også give os mulighed for at screene for lægemidler, der selektivt hæmmer RNA-polymerase, hvilket åbner døren for at udvikle nye lægemidler til behandling af borreliose.Til at begynde med skal du samle cellepillen fra to til fire liter Borreliella burgdorferi RpoC-His10X dyrket i BSKII-medium i et mikroaerofilt miljø til en densitet på to til fire gange 10 konstant pr. Milliliter. Pellet cellerne ved 10.000 gange G i 30 minutter ved fire grader Celsius i 500 ml centrifugalflasker og kassér supernatanten. Derefter resuspenderes cellerne i 30 ml iskold HN-buffer, centrifugeringstrinnet gentages, og supernatanten dekanteres.Mens du arbejder, skal du vedligeholde cellerne, lysatet og de rensede proteiner ved fire grader Celsius eller på is, medmindre de fryser. Opbevar RNA-polymerase i et reducerende miljø ved at tilsætte frisklavet dithiothreitol i en koncentration på to millimolær til hver anvendt buffer og opretholde pH 8,0. Forbered lysat fra Borreliella burgdorferi cellepellet ved hjælp af et kommercielt bakterielt lysesæt.Resuspender pellet i 10 til 15 ml B-PER opløsning uden proteasehæmmer og lad lysen fortsætte i fem minutter på is. Tilsæt en proteasehæmmercocktail til lysisopløsningen og fortsæt med tre runder sonikering. Afklar nu cellelysatet ved centrifugering og filtrering ved hjælp af et 50 ml centrifugerør.Tilsæt koboltsøjleindlæsningsbuffer til opløsningen, hvilket gør det samlede volumen op til 30 ml. Pellet celleaffaldet ved centrifugering ved 20.000 gange G i 30 minutter. Derefter filtreres supernatanten ved hjælp af et 0,45 mikrometer sprøjtefilter, og lysat- og koboltsøjlebufferen fortyndes til et endeligt fortyndingsforhold på 1:10.Der udføres affinitetskromatografi på supernatanten klaret cellelysat ved hjælp af en kobolt- eller nikkelharpikssøjle efter fabrikantens anvisninger. Gem gennemstrømnings-, vaske- og eluerede prøver til analyse. Udskift straks RNA-polymeraseopløsningens bufferopløsning med opbevaringsbufferopløsningen ved hjælp af en bufferudvekslingskolonne efter producentens anvisninger.Derefter koncentreres RNA-polymerasen til en koncentration på 0,2 til 0,4 milligram pr. milliliter ved anvendelse af 10-kilodalton-cutoff centrifugalfilterenheder. Koncentrationen bestemmes ved hjælp af spektrofotometer, og fryselagrene forberedes. Aliquot 20 til 50 mikroliter volumener RNA-polymerasefrysning af lagre i PCR-rør og opbevar RNA-polymerasen ved minus 80 grader Celsius.Forbered arbejdsfladerne, herunder strålingsbænken, for at reducere strålingsforurening. Optø friske RNA-polymerase- og RpoD-lagre på is, og bland alle de optøede frosne lagre grundigt inden pipettering Forbered en masterreaktionsblanding i en separat NTP-blanding til radioaktivt mærkede nukleotider i henhold til de eksperimentelle krav. Fordel kontrolreaktionerne i eksperimentelle sæt i PCR-rør.Efter dispensering af vand, reaktionsblanding, RNA-polymerase og RpoD i udpegede PCR-rør blandes reagenserne ved pipettering. Overfør de forberedte materialer til en strålingsbænk, tilsæt alfa-32P-mærket ATP til NTP, og bland ved blid pipettering. Der tilsættes NTP til rørene indeholdende in vitro-transkriptionsreaktionsblandingerne, og start in vitro-transkriptionsreaktionen ved at tilføje en DNA-skabelon.Reaktionsvolumenet blandes forsigtigt ved pipettering og inkuberer rørene ved 37 grader Celsius i fem minutter i en termocyklisk eller varmeblok. Fjern reaktionerne fra termocykleren eller varmeblokken, og stop in vitro-transkriptionsreaktionerne ved at tilsætte et lige så stort volumen 2X RNA-belastningsfarvestof indeholdende 50% formamid til reaktionsblandingen. Denaturering af enzymerne ved inkubation af reaktioner ved 65 grader Celsius i fem minutter i en termocyklisk eller varmeblok.Ved hjælp af gelelektroforese adskilles det in vitro transkriberede RNA i 10% til 15% TBE urea polyacrylamidgeler ved 180 volt i 30 til 45 minutter. Efter fjernelse af en hvilken som helst del af gelen, der indeholder ikke-inkorporeret radioaktivt mærket ATP, skal du udsætte gelen for phosphoskærmen natten over og afbilde det radioaktivt mærkede RNA ved hjælp af et phosphoscreen-kamera. SDS-PAGE viste tre bånd svarende til tre peptider, RpoC, RpoB og RpoA af RNA-polymerasekompleks.Et peptid på 115 kilodalton, der matchede størrelsen af den MBP-mærkede rekombinante Borreliella burgdorferi RpoD, blev også observeret. Spaltning af proteinblandingen med faktor XA-protease førte til dannelsen af to signifikante produkter, RpoD og MBP. Promotorstedet for Borreliella burgdorferi FLGB blev genereret af PCR.En dobbelt fortynding af RpoD-koncentrationen giver anledning til et lavere niveau af det akkumulerede RNA-produkt. En lav RNA-polymerasekoncentration giver færre RNA-produkter på tværs af RpoD-koncentrationerne. Densitometrisignalet indikerede et lineært forhold mellem mængden af RpoD til stede i reaktionen i begge eksperimenter.RNA-polymeraseenzymatisk aktivitet er følsom over for en række faktorer under oprensning, opbevaring og eksperimentel proces. Frisk enzym og reagenser sammen med god planlægning giver den bedst mulige chance for succes i denne protokol.

Research

JoVE Journal

Immunology and Infection

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitative<em&gt; In vitro</em&gt; Assay til måling neutrofiladhæsion til Aktiverede primære humane mikrovaskulære endotelceller under statiske forhold

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Oprensning af<em&gt; Plasmodium falciparum</em&gt; Merozoites Til brug i opsoniserende antistofanalyser

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Assays til at studere rollen, som Vitronectin i bakteriel adhæsion og Serum modstand

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In vitro- metoder til sammenligning af målbinding og CDC-induktion mellem terapeutiske antistoffer: applikationer i biosimilaritetsanalyse

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

Screening Assays for at karakterisere romanen endotel regulerende myndigheder involveret i inflammatorisk reaktion

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

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

Høj overførselshastighed i in vitro-vurdering af Latency vende agenter på HIV transskription og splejsning

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 ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Mønster-udløst oxidativ Burst og Seedling væksthæmning assays i Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Skræddersy in vivo cytotoksiske assays til at studere immun dominans i tumor specifikke CD8+ T- celle respons

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Analyse af interaktioner mellem Endobiotika og humant tarm mikrobiota ved hjælp af in vitro-bade fermenterings systemer

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...