Name: a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry
Uploaded: 2013-04-09T13:00:00
Description: Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com

Forfatterne vil gerne takke Nancy Mora for hendes teknisk bistand. Dette arbejde blev finansieret af forskningsmidler 48.573-M og 168.098 fra Consejo Nacional de Ciencia y Tecnologia, Mexico, og af tilskud IN212308 og IN205311-2 fra Dirección General de asuntos del Personal Academico, Universidad Nacional Autonoma de Mexico, Mexico.

1. Isolering af Neutrofiler (PMN) fra humant blod <ol><li> Brug 20 ml humant blod med heparin (10 U / ml) som antikoagulant. Blod blev indsamlet fra raske, voksne frivillige ved venopuncture. Alle forsøg blev udført under godkendelse af bioetiske komité ved Instituto de Investigaciones Biomédicas - UNAM. </li><li> Put 2 ml 6% dextran T500 i PBS i en 15 ml konisk centrifugeglas, og der tilsættes 10 ml blod. Bland ved at vende røret to eller tre gange og lad det sidde i 45 minutter for at tillade erytrocytsænknin...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+neutrophil+apoptosis:+new+players+enter+the+game.">Regulating neutrophil apoptosis: new players enter the game.</a> Trends Immunol. 32, 117-124 (2011).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+and+NF-&#954;B+in+T+cell+activation:+life+at+the+edge.">Death and NF-&#954;B in T cell activation: life at the edge.</a> Mol. Cell. 11, 551-552 (2003).</li><li>Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., Franzoso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+JNK+signaling+to+NF-&#954;B:+a+key+to+survival.">Linking JNK signaling to NF-&#954;B: a key to survival.</a> J. 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Meth. 320, 104-118 (2007).</li><li>Garcia-Garcia, E., Nieto-Castaneda, G., Ruiz-Saldana, M., Mora, N., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc&#947;RIIA+and+Fc&#947;RIIIB+mediate+nuclear+factor+activation+through+separate+signaling+pathways+in+human+neuthophils.">Fc&#947;RIIA and Fc&#947;RIIIB mediate nuclear factor activation through separate signaling pathways in human neuthophils.</a> J. Immunol. 182, 4547-4556 (2009).</li></ol>

Oprensningen her beskrevne fremgangsmåde tillader isolering af ikke-stimulerede neutrofiler (PMN) med renhed på over 95% (vurderet ved mikroskopisk observation), på kort tid. Sommetider neutrofiler kan være forurenet med erythrocyter hvis sidstnævnte ikke lyseres fuldstændigt. Dette påvirker normalt ikke teknikken, idet erythrocytter og PMN let kan skelnes som distinkte cellepopulationer ved flowcytometri. Isolerede PMN kan derefter stimuleres ved tværbinding af bestemte receptorer med specifikke monoklonale ant...

Neutrofiler er de mest udbredte leukocytter i perifert blod 1. Under inflammation og infektion neutrofiler er de første celler til at vises på det angrebne sted, hvor de fungerer som den første linje i forsvaret 2. Neutrofiler har flere antimikrobielle mekanismer 3 herunder fagocytose, produktion af reaktive ilt arter, frigivelse af lytiske enzymer ved degranulering, og produktion af proinflammatoriske cytokiner 4,5. Neutrofiler er kortlivede celler, der bliver hurtigt aktiveres ved hjælp af signalering fra forskellige celleoverfladereceptorer. Selv neutrofiler er blevet overvejet terminale celler på grund af deres korte levetid og fordi de undergår apoptose, medmindre aktiveret under den inflammatoriske proces 6, er det nu klart, at de også kan ændre deres fænotype ved at ændre niveauet for transkription af bestemte gener. Produktion af cytokiner 5 og inhibering af apoptose 7,8 er to important aktivering-afhængige cellefunktioner reguleret på niveauet for transkription i neutrofiler. Nukleare faktor KB (NF-KB) deltager i transkriptionel kontrol af cytokinproduktion 4 og ved regulering af celleoverlevelse og apoptose 9-11 i forskellige celletyper. De signalveje, der fører til kernefaktor aktivering sædvanligvis undersøgt ved reportergenassays eller ved elektroforetisk mobilitet shift assays (EMSA). Men da neutrofiler er kortlivede celler, kan studiet af transkriptionelt regulerede responser i disse celler ikke udføres med reportergenassays, da der ikke findes effektive teknikker til neutrofil transfektion. EMSA assays er blevet anvendt i neutrofiler til at udforske kernefaktor aktivering 12,13, men denne metode er kompliceret og dyr, fordi den involverer anvendelsen af radioaktive materialer. Nucleofection er en anden teknik, der har været anvendt successfully at transficere monocytter 14. I det mindste i teorien, kunne kernefaktor aktivering påvises i neutrofiler efter transfektion (trods lav effektivitet). Imidlertid vil denne teknik være dyrere, tidskrævende og sandsynligvis mindre kvantitativ. Mikroskopisk analyse af immunofarvede celler kunne også anvendes til påvisning af nukleare faktorer i kernen. Vi har faktisk opdaget NF-KB translokation til kernen på denne måde 15. Desværre er denne teknik er også tidskrævende, mindre kvantitativ, og med forbehold observatørens bias. Her præsenteres en simpel og effektiv fremgangsmåde, der tillader detektering og kvantificering af nukleare faktorer i isoleret og immunolabeled kerner ved flowcytometri. Vi beskriver teknikker til isolering af neutrofiler fra humant perifert blod, stimulere disse celler via integriner eller Fc-receptorer med anti-receptor-antistoffer, isolere og immunolabel kerner, og analysere kerner ved hjælp af flowcytometri (Figur 1). Fremgangsmåden er med succes blevet anvendt til påvisning af NF-KB 15 og Elk-1 16 nukleare faktorer i neutrofil kerner. Følsomheden af ​​denne fremgangsmåde tillader detektion af små ændringer i nukleare faktor niveauer i kernen. Denne fremgangsmåde kan også anvendes til at analysere niveauet af transkriptionsfaktorer i kerner fra andre celletyper.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

Neutrofiler er de mest udbredte leukocytter i perifert blod. Disse celler er de første til at blive vist på steder med inflammation og infektion, og blev dermed den første linje i forsvaret mod invaderende mikroorganismer. Neutrofiler har vigtige antimikrobielle funktioner såsom fagocytose, frigivelse af lytiske enzymer, og produktionen af ​​reaktive oxygenspecies. Ud over disse vigtige forsvar funktioner, udfører neutrofiler andre opgaver som reaktion på infektion, såsom produktion af proinflammatoriske cytokiner og inhibering af apoptose. Cytokiner rekruttere andre leukocytter, der hjælper klare infektionen, og inhibering af apoptose tillader neutrofil at leve længere på stedet for infektion. Disse funktioner er reguleret på niveauet for transkription. Men da neutrofiler er kortlivede celler, kan studiet af transkriptionelt regulerede responser i disse celler ikke udføres med konventionelle reportergen metoder, da der ikke er nogen effektifaktor teknikker til neutrofil transfektion. Her præsenteres en simpel og effektiv fremgangsmåde, der tillader detektering og kvantificering af nukleare faktorer i isoleret og immunolabeled kerner ved flowcytometri. Vi beskriver teknikker til at isolere rene neutrofiler fra humant perifert blod, stimulere disse celler med anti-receptor-antistoffer, isolere og immunolabel kerner, og analysere kerner ved flowcytometri. Fremgangsmåden er med succes blevet anvendt til påvisning af NF-KB og Elk-1 nukleare faktorer i kerner fra neutrofiler og andre celletyper. Således er denne fremgangsmåde repræsenterer en indstilling til analyse af aktivering af transkriptionsfaktorer i isolerede kerner fra en række celletyper.

Oprensningen beskrevet her giver normalt ikke-stimulerede neutrofiler (PMN) med renhed på over 95% (figur 1A). Isolerede PMN kan derefter stimuleres ved tværbinding af bestemte receptorer med specifikke monoklonale antistoffer. Vi har stimuleret PMN via Fc-receptorer og integriner (figur 1B). Når stimuleres, er PMN lyseres og kerner er isoleret med høje udbytter. Kerner derefter immunolabeled for en bestemt nukleær faktor, såsom nukleare faktor KB (NF-KB), med specifikke antistoff...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com 

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry (Video) | JoVE 

Neutrofiler er de mest udbredte leukocytter i blodet. Neutrofiler besidder transkriptionelt regulerede funktioner, såsom produktion af proinflammatoriske cytokiner og inhibering af apoptose. Disse funktioner kan undersøges med fremgangsmåden præsenteret her, som muliggør påvisning og kvantificering af nukleare faktorer ved flowcytometri i isolerede kerner

En enkel og effektiv metode til at påvise Nuclear Factor Aktivering i humane neutrofiler ved flowcytometri

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A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

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