Name: isolation, transfection, and culture of primary human monocytes
Uploaded: 2019-12-16T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Forfatterne vil gjerne takke HIV Clinical/tumor Biorepository Core for å gi pasienten prøver og Cellular immunologi metabolisme Core for å gi flyt flowcytometri analyse. Dette prosjektet ble finansiert av NIH P20GM121288 og P30GM114732.

Alle metodene som er beskrevet nedenfor er godkjent av Louisiana State University Health Sciences Center New Orleans institusjonelle gjennomgang Board. Alt blod ble samlet inn etter innhente informert samtykke.
Merk: hele prosedyren utføres under sterile forhold i et biosafety nivå 2 (BSL2) anlegg, slik at forsiktighet brukes til å håndtere biologiske materialer. Spesielt er hvert trinn utføres ved hjelp av sterile teknikker under et biosafety kabinett. Etter hvert trinn som involverer bl...

<ol>
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J., 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=Macrophage+M1/M2+polarization+dynamically+adapts+to+changes+in+cytokine+microenvironments+in+Cryptococcus+neoformans+infection.">Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection.</a> MBio. 4 (3), e00264 (2013).</li><li>Raggi, F., 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=Regulation+of+Human+Macrophage+M1-M2+Polarization+Balance+by+Hypoxia+and+the+Triggering+Receptor+Expressed+on+Myeloid+Cells-1.">Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.</a> Frontiers in Immunology. 8, 1097 (2017).</li><li>Van Overmeire, E., 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=M-CSF+and+GM-CSF+Receptor+Signaling+Differentially+Regulate+Monocyte+Maturation+and+Macrophage+Polarization+in+the+Tumor+Microenvironment.">M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.</a> Cancer Research. 76 (1), 35-42 (2016).</li><li>Vogel, D. Y., 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=Human+macrophage+polarization+in+vitro:+maturation+and+activation+methods+compared.">Human macrophage polarization in vitro: maturation and activation methods compared.</a> Immunobiology. 219 (9), 695-703 (2014).</li><li>Almeida, M., Cordero, M., Almeida, J., Orfao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+subsets+of+peripheral+blood+dendritic+cells+show+distinct+phenotypic+and+functional+abnormalities+in+HIV-1+infection.">Different subsets of peripheral blood dendritic cells show distinct phenotypic and functional abnormalities in HIV-1 infection.</a> AIDS. 19 (3), 261-271 (2005).</li><li>Ciesek, S., 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=Impaired+TRAIL-dependent+cytotoxicity+of+CD1c-positive+dendritic+cells+in+chronic+hepatitis+C+virus+infection.">Impaired TRAIL-dependent cytotoxicity of CD1c-positive dendritic cells in chronic hepatitis C virus infection.</a> Journal of Viral Hepatitis. 15 (3), 200-211 (2008).</li><li>Granelli-Piperno, A., Golebiowska, A., Trumpfheller, C., Siegal, F. P., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1-infected+monocyte-derived+dendritic+cells+do+not+undergo+maturation+but+can+elicit+IL-10+production+and+T+cell+regulation.">HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (20), 7669-7674 (2004).</li><li>Hearps, A. C., 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=HIV+infection+induces+age-related+changes+to+monocytes+and+innate+immune+activation+in+young+men+that+persist+despite+combination+antiretroviral+therapy.">HIV infection induces age-related changes to monocytes and innate immune activation in young men that persist despite combination antiretroviral therapy.</a> AIDS. 26 (7), 843-853 (2012).</li><li>Heggelund, L., 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=Stimulation+of+toll-like+receptor+2+in+mononuclear+cells+from+HIV-infected+patients+induces+chemokine+responses:+possible+pathogenic+consequences.">Stimulation of toll-like receptor 2 in mononuclear cells from HIV-infected patients induces chemokine responses: possible pathogenic consequences.</a> Clinical and Experimental Immunology. 138 (1), 116-121 (2004).</li><li>Hernandez, J. C., 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=Up-regulation+of+TLR2+and+TLR4+in+dendritic+cells+in+response+to+HIV+type+1+and+coinfection+with+opportunistic+pathogens.">Up-regulation of TLR2 and TLR4 in dendritic cells in response to HIV type 1 and coinfection with opportunistic pathogens.</a> AIDS Research and Human Retroviruses. 27 (10), 1099-1109 (2011).</li><li>Hernandez, J. C., Latz, E., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+induces+the+first+signal+to+activate+the+NLRP3+inflammasome+in+monocyte-derived+macrophages.">HIV-1 induces the first signal to activate the NLRP3 inflammasome in monocyte-derived macrophages.</a> Intervirology. 57 (1), 36-42 (2014).</li><li>Low, H. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Impairment+of+Myeloid+Dendritic+Cells+during+Advanced+Stage+of+HIV-1+Infection:+Role+of+Factors+Regulating+Cytokine+Signaling.">Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling.</a> PLoS ONE. 10 (10), e0140852 (2015).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+negative+regulators+of+cytokine+signaling+during+chronic+HIV+disease+cause+functionally+exhausted+state+of+dendritic+cells.">Increased expression of negative regulators of cytokine signaling during chronic HIV disease cause functionally exhausted state of dendritic cells.</a> Cytokine. 91, 118-123 (2017).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Li, H., Jiang, T., Li, M. Q., Zheng, X. L., Zhao, G. 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J., Cohen, E. A., Xiong, S. D., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-1236+inhibits+HIV-1+infection+of+monocytes+by+repressing+translation+of+cellular+factor+VprBP.">miRNA-1236 inhibits HIV-1 infection of monocytes by repressing translation of cellular factor VprBP.</a> PLoS ONE. 9 (6), e99535 (2014).</li><li>Riess, M., 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=Interferons+Induce+Expression+of+SAMHD1+in+Monocytes+through+Down-regulation+of+miR-181a+and+miR-30a.">Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.</a> Journal of Biological Chemistry. 292 (1), 264-277 (2017).</li><li>Buchacher, T., Ohradanova-Repic, A., Stockinger, H., Fischer, M. 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Den presenterte protokollen demonstrerer bruk av primær celler fra HIV-smittede som en modell for å studere monocytter og makrofager. HIV+ pasienter gjennomgår cART leve med infeksjon i flere år og kan også ha andre co-infeksjoner knyttet en kompromittert immunsystem. Å studere immunomodulation i nærvær av HIV kronisk infeksjon, celler ble høstet fra pasienter direkte. Som miRNAs har vist å spille store roller i celle utvikling og differensiering, fokuserer protokollen på evnen til å manipulere miR...

Humant immunsviktvirus-1 (HIV-1) infeksjon forårsaker alvorlig immun dysfunksjon, som kan føre til opportunistiske infeksjoner og ervervet immunsviktsyndrom (AIDS). Selv om HIV-smittede pasienter under cART er preget av lav viral belastninger og normal CD4+ T Cell teller, funksjon av immunsystemet kan bli kompromittert i disse individene, som fører til en dysfunksjonelle immunrespons som har vært knyttet til en økt risiko for å utvikle kreft1. Mekanismene for immun dysfunksjon i HIV-pasienter i handlevognen fortsatt i stor grad ukjent. Derfor karakteriserer pasient-avledet immunceller og gransker deres biologi og funksjon er en kritisk komponent i dagens HIV-forskning.
Monocytter og makrofager er sentrale regulatorer av immunresponser og spille grunnleggende roller i HIV-smitte2,3,4,5. Heterogen og plast i naturen, makrofager kan grovt klassifiseres i klassisk aktivert (M1) eller alternativt aktivert (M2). Mens denne generelle klassifiseringen er nødvendig når du setter opp eksperimentelle vilkår, polarisering status av makrofager kan reverseres av en rekke cytokiner6,7,8,9. Selv om flere studier har undersøkt virkningene av HIV-smitte på monocytter og dendrittiske celler, molekylære detaljer om monocytt-mediert svar er i stor grad ukjent6,7,10,11,12,13,14,15,16,17,18,19. Blant de faktorene som er involvert i immun celle regulering og funksjon, microRNAs (miRNAs), kort ikke-koding RNAs at post-transcriptionally regulere genuttrykk, har vist seg å spille en viktig rolle i sammenheng med store cellulære veier (dvs. vekst, differensiering, utvikling, og apoptose)20. Disse molekylene har blitt beskrevet som viktige regulatorer av transkripsjon faktorer avgjørende for å diktere funksjonell polarisering av makrofager21. Den potensielle rollen til miRNAs i monocytter fra HIV-infiserte individer gjennomgår handlevognen har blitt undersøkt, men fremgangen i feltet krever mye mer arbeid22,23,24,25,26. Dette papiret diskuterer en optimalisert metode for å transfect miRNAs og siRNAs i primære menneskelige monocytter fra HIV-infiserte pasienter og kontroller.
Denne protokollen er avhengig av kommersielt tilgjengelige reagenser og sett, som kontinuitet i den tekniske prosedyren bidrar til å eliminere unødvendige eksperimentelle variabler når du arbeider med kliniske prøver. Likevel gir metoden viktige tips (dvs. antall celler belagt eller kort inkubasjons med serum-frie medier for å fremme overholdelse av celler til platen). I tillegg er polarisering forholdene som brukes i denne protokollen avledet fra publisert arbeid27,28,29.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD Horizon</td>
			<td>563794</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD14 PerCP</td>
			<td>Invitrogen</td>
			<td>46-0149-42</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD163 BV711</td>
			<td>BD Horizon</td>
			<td>563889</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD209 BV421</td>
			<td>BD Horizon</td>
			<td>564127</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD80 FITC</td>
			<td>BD Horizon</td>
			<td>557226</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD83 APC</td>
			<td>BD Horizon</td>
			<td>551073</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>EIF4EBP1 mAb</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>EIF4EBP1 siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-29594</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum Defined Heat Inactivated</td>
			<td>Hyclone</td>
			<td>SH30070.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>B43618</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH mAb</td>
			<td>Santa Cruz</td>
			<td>SC-47724</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>HuFcR Binding Inhibitor</td>
			<td>eBiosciences</td>
			<td>14-9161-73</td>
			<td>Flow cytometry- blocking buffer</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>B16406</td>
			<td>Software to analyze flow cytometry data</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O55:B5</td>
			<td>Sigma</td>
			<td>L4524</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA microRNA Mimic hsa-miR-146a-5p</td>
			<td>Qiagen</td>
			<td>YM00472124</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION miRNA Negative Control</td>
			<td>Sigma</td>
			<td>HMC0002</td>
			<td>Scrambled miRNA conjugated with a near infrared dye</td>
		</tr>
		<tr>
			<td>Nunc 35mm Cell Culture Dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>R&#38;D Systems</td>
			<td>216-MC-025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrambled Control siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-37007</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Blue Transfection Reagent Kit</td>
			<td>Lipocalyx</td>
			<td>VB-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1 Cell Proliferation Reagent</td>
			<td>Roche</td>
			<td>5015944001</td>
			<td>Colorimetric assay to assess cell viability</td>
		</tr>
	</tbody>
</table>

isolation, transfection, and culture of primary human monocytes

Humant immunsviktvirus (HIV) er fortsatt en stor helse bekymring til tross for innføringen av kombinert antiretroviral behandling (cART) på midten av 1990-tallet. Mens antiretroviral behandling effektivt senker systemisk viral belastning og gjenoppretter normal CD4+ T celle tellinger, betyr det ikke Rekonstituer en helt funksjonell immunsystem. En dysfunksjonelle immunsystem i HIV-infiserte individer under cART kan være preget av immun aktivering, tidlig aldring av immunceller, eller vedvarende betennelse. Disse forholdene, sammen med komorbide faktorer forbundet med HIV-smitte, legger kompleksiteten til sykdommen, som ikke kan lett gjengis i cellulære og dyremodeller. For å undersøke den molekylære hendelser underliggende immun dysfunksjon hos disse pasientene, et system til kultur og manipulere menneskelig primære monocytter in vitro er presentert her. Nærmere bestemt, tillater protokollen for kultur og transfeksjoner av primære CD14+ monocytter innhentet fra HIV-infiserte individer gjennomgår cART så vel som fra HIV-negative kontroller. Metoden innebærer isolasjon, kultur, og transfeksjoner av monocytter og monocytt-avledede makrofager. Mens kommersielt tilgjengelige kits og reagenser er ansatt, gir protokollen viktige tips og optimaliserte vilkår for vellykket overholdelse og transfeksjoner av monocytter med miRNA og hemmere, så vel som med siRNAs.

Ved hjelp av prosedyren beskrevet, primære menneskelige monocytter fra HIV-smittede individer og sunne donorer ble isolert. Alle data som presenteres her ble innhentet fra HIV+ gjennomgår cART med lav (< 20 eksemplarer/ml) eller undetectable viral belastninger og normal CD4+ T Cell teller. Umiddelbart etter isolasjon, celler ble farget, og flyt flowcytometri ble utført for å bekrefte renheten av celle populasjoner. Resultatene viste at > 97% av cellene farget pos...

Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Isolation, Transfection, and Culture of Primary Human Monocytes

Presentert her er en optimalisert protokoll for å isolere, dyrking, transfecting, og differensiere menneskets primære monocytter fra HIV-smittede individer og sunne kontroller.

Isolasjon, Transfeksjoner, og kultur for Primary Human monocytter

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

HIV-infeksjon forårsaker immundysfunksjon selv hos personer som gjennomgår antiretroviral terapi uten påviselig virus. Denne metoden gjør det mulig for studiet av monocyttfunksjon som er viktige regulatorer av immunresponsen. Vi har optimalisert denne teknikken for isolasjon, kultur og transfection av menneskelige primære monocytter.Vi bruker denne metoden til å forstå de molekylære mekanismene for immundysfunksjon hos HIV-infiserte individer, men det kan brukes på andre immunstudier som involverer monocytter. Hvis dette er første gang du arbeider med menneskelig blod, husk å følge de riktige biosikkerhetsprotokollene og å behandle alle prøvene som muligens smittsomt materiale. Etter å ha samlet 40 milliliter friskt menneskelig fullblod i fire 10 milliliter EDTA vakuumrør, bruk steril teknikk for å overføre alt blodet til et enkelt 50 milliliter konisk propylenrør i et biosikkerhetsskap.Etter produsentens instruksjoner fra et valgt menneskelig monocyttisolasjonssett, legg til to milliliter monocyttisolasjonscocktail fra settet til blodrøret og virvle de magnetiske perlene fra settet i 30 sekunder. Tilsett to milliliter perler til blodet og bruk en 25 milliliter serologisk pipette for å forsiktig blande perlene med blodet. Etter fem minutter ved romtemperatur, dele blodet likt mellom fire 50 milliliter rør og tilsett 30 milliliter steril PBS supplert med en millimolar EDTA til hvert rør.Bland igjen med en plast 25 milliliter serologisk pipette og plasser rørene i magnetiske holdere. Etter 10 minutter, bruk en pipette til å trekke opp innholdet fra midten av hvert rør, og vær forsiktig så du ikke aspirerer mer enn en milliliter røde blodlegemer og dispenserer rørinnholdet i en av fire nye 50 milliliter rør. Tilsett 500 ekstra mikroliter av de vortexed magnetiske perlene til hvert rør og bland forsiktig celleløsningen.Plasser rørene tilbake i magnetholderne i fem minutter før du forsiktig overfører innholdet fra midten av hvert rør til ett av fire nye 50 milliliter rør. Når alle cellesuspensjonene er overført, plasserer du hvert nytt 50 milliliter rør i magnetholderne i fem minutter før du forsiktig overfører rørinnholdet til et tredje sett med fire nye 50 milliliter rør. Deretter samle cellene ved sentrifugering og resuspend alle fire celle pellets i totalt 10 milliliter steril PBS for telling.Etter telling, resuspend de isolerte monocytter på en gang 10 til sjette celler per milliliter på 37 grader Celsius serum-fri RPMI 1640 medium supplert med antibiotika og plate en milliliter av resuspended celler i 35 millimeter plater i et biosikkerhetskap. Plasser deretter platene i cellekulturinkubatoren i 30 til 60 minutter for å la cellene holde seg til brønnbunnene. For å prime for makrofager med en M1-lignende fenotype, tilsett 100 mikroliter føtal storfe serum som inneholder 25 nanogram per milliliter GM-CSF til hver plate.For å prime for makrofager med en M2-lignende fenotype, legg til 100 mikroliter fostervin som inneholder 50 nanogram per milliliter M-CSF til hver plate. For monocytttransfeksjon med microRNA etterligner, hemmere eller en liten forstyrrende RNA, etter produsentens protokoll for transfeksjonssettet, fortynne de valgte RNA-ene i bufferen til en endelig konsentrasjon på 1,83 mikrorør i 10 mikroliter fortynnet etterligne eller hemmer per transfeksjon av en gang 10 til femte celler volum. For å forberede transfection reagensen, tilsett en mikroliter av den medfølgende polymeren til et 1,5 milliliter mikrocentrifugerør og legg umiddelbart til 90 mikroliter av den kit-gitte bufferen for å oppnå totalt 91 mikroliter reagens per transfeksjon.Vortex regenten i tre til fem sekunder og pipette 90 mikroliter av transfection løsningen inn i røret som inneholder 10 mikroliter fortynnet RNA. Etter mild blanding, inkuber oppløsningen i 15 minutter ved romtemperatur før du legger til 100 mikroliter transinfeksjonskompleks til en brønn av celler. Etter fire timer ved 37 grader Celsius, erstatt mediet med tre milliliter komplett medium som inneholder riktig vekstfaktor.På dag seks av kulturen, erstatte kultur supernativa fra M1 kulturer med tre milliliter av medium supplert med fosterbovin serum, antibiotika, lipopolysakkarid, og interferon gamma og erstatte supernativa fra M2 kulturer med medium supplert med fosterbovin serum, antibiotika, M-CSF, og interleukin-4. Etter 24 timers kultur, vask hver plate av aktiverte makrofager to ganger med frisk PBS per vask. For RNA- og proteinanalyser lyser de vedlagte cellene direkte i platene for å tillate innsamling av det frigjorte celleinnholdet for deres analyse.For flow cytometrisk analyse, koble cellene med to millimolar EDTA i PBS i 10 minutter ved 37 grader Celsius før forsiktig skraping cellene fra platebunnen for å tillate deres samling i 1,5 milliliter mikrocentrifuge rør for deres analyse. Her vises representative histogrammer av M1-aktivert kontroll og pasientavledede celler med økte nivåer av CD80- og CD83- og M2-aktiverte celler med økte nivåer av CD163 og CD209 sammenlignet med ikke-aktiverte T0-celler. Interessant, CD80 og CD83 syntes å være mer høyt uttrykt i kontroll-avledede celler sammenlignet med HIV-avledede celler.Transfection av nyinnsamlede monocytter med en kryptert nær-infrarød merket microRNA resulterer i en større enn 90% effektivitet av transfection som bestemmes av strømningscytometri og konokal mikroskopi. I tillegg reduserer transfection ikke levedyktigheten til pasientavledede eller kontrollceller betydelig uavhengig av cellemodning eller transinfeksjonsforholdene. Transinfeksjon resulterer i en effektiv nedregulering av målgenet ved transinfeksjon med den spesifikke lille forstyrrende RNA på både dag én og dag fire etterisolering.Videre viser celler som er transfisert med microRNA-etterligne en 48 til 72 ganger økning i microRNA-uttrykk over uoversvevete celler, mens alle transfeksjonskontrollene ikke viser merkbare endringer. Antall celler belagt er avgjørende for å overleve. Vi anbefaler en million celler per 35 millimeter rett.Plating i serumfritt medium vil også forbedre cellevedlegget sterkt. Celler oppnådd med denne metoden kan behandles med cytokiner eller vekstfaktorer for å studere differensialresponser hos pasienter av interesse og sunne kontroller.

isolation-transfection-and-culture-of-primary-human-monocytes

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generering av Organotypic Raft Cultures fra primære humane keratinocytter

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolering av humane monocytter ved å dobbelt gradientsentrifugering og deres differensiering til Makrofager i teflonbelagt cellekultur Vesker

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

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.

Svært effektiv Transfeksjon menneskerettighets THP-1 Makrofager av nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolasjon og intravenøs injeksjon av murine Bone Marrow Avledet Monocytter

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymfocyttisolasjonsmetoder fra menneskelig hud for fenotypeanalyser og<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Levende Imaging av Antifungal aktivitet av humane primære nøytrofile granulocytter og monocytter i Response til<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Liten RNA Transfeksjon i Primær Menneskelig Th17 celler av Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolasjon og In Vitro kultur Murine og menneskelig Alveolar makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Mus - og menneske-avledet primære mage Epithelial Monolayer kultur for studier av gjenfødelse

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...