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

Gli autori desiderano ringraziare il nucleo del biorepository clinico/tumorale dell'HIV per aver fornito campioni di pazienti e il nucleo del metabolismo dell'immunologia cellulare per la fornitura dell'analisi della citometria del flusso. Questo progetto è stato finanziato da NIH P20GM121288 e P30GM114732.

Tutti i metodi descritti di seguito sono stati approvati dal Louisiana State University Health Sciences Center New Orleans Institutional Review Board. Tutto il sangue è stato raccolto dopo aver ottenuto il consenso informato.
NOTA: L'intera procedura viene eseguita in condizioni sterili in un impianto di biosicurezza di livello 2 (BSL2) in modo che venga utilizzata cautela per la gestione di materiali biologici. In particolare, ogni passo viene eseguita utilizzando tecniche sterili sotto un a...

<ol>
	<li>Slim, J., Saling, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Management+of+Inflammation+in+the+HIV+Population.">A Review of Management of Inflammation in the HIV Population.</a> Biomedical Research International. 2016, 3420638 (2016).</li><li>Herskovitz, J., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+the+Macrophage:+From+Cell+Reservoirs+to+Drug+Delivery+to+Viral+Eradication.">HIV and the Macrophage: From Cell Reservoirs to Drug Delivery to Viral Eradication.</a> Journal of Neuroimmune Pharmacology. 14 (1), 52-67 (2019).</li><li>Machado Andrade, V., Stevenson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+Viral+Factors+Influencing+Interplay+between+the+Macrophage+and+HIV-1.">Host and Viral Factors Influencing Interplay between the Macrophage and HIV-1.</a> Journal of Neuroimmune Pharmacology. 14 (1), 33-43 (2019).</li><li>Merino, K. M., Allers, C., Didier, E. S., Kuroda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Monocyte/Macrophages+during+HIV/SIV+Infection+in+Adult+and+Pediatric+Acquired+Immune+Deficiency+Syndrome.">Role of Monocyte/Macrophages during HIV/SIV Infection in Adult and Pediatric Acquired Immune Deficiency Syndrome.</a> Frontiers in Immunology. 8, 1693 (2017).</li><li>Wacleche, V. S., Tremblay, C. L., Routy, J. P., Ancuta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biology+of+Monocytes+and+Dendritic+Cells:+Contribution+to+HIV+Pathogenesis.">The Biology of Monocytes and Dendritic Cells: Contribution to HIV Pathogenesis.</a> Viruses. 10 (2), (2018).</li><li>Davis, M. 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. Z., 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=TLR8+regulation+of+LILRA3+in+monocytes+is+abrogated+in+human+immunodeficiency+virus+infection+and+correlates+to+CD4+counts+and+virus+loads.">TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads.</a> Retrovirology. 13, 15 (2016).</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=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. B., Weber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M2+Polarization+of+Human+Macrophages+Favors+Survival+of+the+Intracellular+Pathogen+Chlamydia+pneumoniae.">M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.</a> PLoS ONE. 10 (11), e0143593 (2015).</li><li>Jaguin, M., Houlbert, N., Fardel, O., Lecureur, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+profiles+of+human+M-CSF-generated+macrophages+and+comparison+of+M1-markers+in+classically+activated+macrophages+from+GM-CSF+and+M-CSF+origin.">Polarization profiles of human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin.</a> Cellular Immunology. 281 (1), 51-61 (2013).</li><li>Lacey, D. 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=Defining+GM-CSF-+and+macrophage-CSF-dependent+macrophage+responses+by+in+vitro+models.">Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models.</a> Journal of Immunology. 188 (11), 5752-5765 (2012).</li><li>Tarique, A. 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=Phenotypic,+functional,+and+plasticity+features+of+classical+and+alternatively+activated+human+macrophages.">Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.</a> American Journal of Respiratory Cell and Molecular Biology. 53 (5), 676-688 (2015).</li></ol>

Il protocollo presentato dimostra l'uso di cellule primarie di soggetti infettati da HIV come modello per studiare monociti e macrofagi. HIV- I pazienti sottoposti a cART vivono con infezione per più anni e possono anche avere altre co-infezioni correlate a un sistema immunitario compromesso. Per studiare l'immunomodulazione in presenza di infezione cronica da HIV, le cellule sono state raccolte direttamente dai pazienti. Poiché i miRNA hanno dimostrato di svolgere un ruolo importante nello sviluppo cellular...

L'infezione da virus immunodeficienza umana-1 (HIV-1) causa una grave disfunzione immunitaria, che può portare a infezioni opportunistiche e sindrome da immunodeficienza acquisita (AIDS). Anche se i pazienti affetti da HIV sottoposti a cART sono caratterizzati da bassi carichi virali e normali conteggi di cellule CD4- T, il funzionamento del sistema immunitario può essere compromesso in questi individui, portando ad una risposta immunitaria disfunzionale che è stata collegata ad un aumento del rischio di sviluppare il cancro1. I meccanismi della disfunzione immunitaria nei pazienti affetti da HIV su cART rimangono in gran parte sconosciuti. Pertanto, caratterizzare le cellule immunitarie derivate dal paziente e studiare la loro biologia e funzione è una componente fondamentale dell'attuale ricerca sull'HIV.
Monociti e macrofagi sono regolatori chiave delle risposte immunitarie e svolgono un ruolo fondamentale nell'infezione da HIV2,3,4,5. Eterogenei e di natura plastica, i macrofagi possono essere ampiamente classificati in attivazione classica (M1) o in alternativa attivati (M2). Mentre questa classificazione generale è necessaria quando si impostano condizioni sperimentali, lo stato di polarizzazione dei macrofagi può essere invertito da una varietà di citochine6,7,8,9. Anche se diversi studi hanno studiato gli effetti dell'infezione da HIV su monociti e cellule dendritiche, i dettagli molecolari delle risposte mediate da monociti sono in gran parte sconosciuti6,7,10,11,12,13,14,15,16,17,18,19. Tra i fattori coinvolti nella regolazione e nella funzione delle cellule immunitarie, i microRNA (miRNA), i brevi RNA non codificanti che regolano post-trascrizione l'espressione genica, hanno dimostrato di svolgere un ruolo importante nel contesto delle principali vie cellulari (cioè, crescita, differenziazione, sviluppo e apoptosi)20. Queste molecole sono state descritte come importanti regolatori di fattori di trascrizione essenziali per dettare la polarizzazione funzionale dei macrofagi21. È stato studiato il ruolo potenziale dei miRNA nei monociti di individui affetti da HIV sottoposti a cART, ma i progressi nel campo richiedono molto più lavoro22,23,24,25,26. Questo documento illustra un metodo ottimizzato per trasfect miRNA e siRNA in monociti umani primari da pazienti e controlli infettati da HIV.
Questo protocollo si basa su reagenti e kit disponibili in commercio, poiché la continuità nella procedura tecnica consente di eliminare le variabili sperimentali non necessarie quando si lavora con campioni clinici. Ciò nonostante, il metodo fornisce suggerimenti importanti (cioè il numero di cellule placcate o una breve incubazione con supporti senza siero per promuovere l'aderenza delle cellule alla piastra). Inoltre, le condizioni di polarizzazione utilizzate in questo protocollo derivano dal lavoro pubblicato27,28,29.

<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

Il virus dell'immunodeficienza umana (HIV) rimane una delle principali preoccupazioni per la salute, nonostante l'introduzione della terapia antiretrovirale combinata (cART) a metà degli anni '90. Mentre la terapia antiretrovirale abbassa in modo efficiente il carico virale sistemico e ripristina il normale CD4- conteggi di cellule T, non ricostituisce un sistema immunitario completamente funzionale. Un sistema immunitario disfunzionale in individui infettati da HIV sottoposti a cART può essere caratterizzato da attivazione immunitaria, invecchiamento precoce delle cellule immunitarie o infiammazione persistente. Queste condizioni, insieme ai fattori comorbidi associati all'infezione da HIV, aggiungono complessità alla malattia, che non può essere facilmente riprodotta nei modelli cellulari e animali. Per studiare gli eventi molecolari alla base della disfunzione immunitaria in questi pazienti, viene presentato un sistema per la coltura e la manipolazione dei monociti primari umani in vitro. In particolare, il protocollo consente la coltura e la trasfezione del CD14 primario- monociti ottenuti da individui affetti da HIV sottoposti a cART e da controlli HIV-negativi. Il metodo prevede l'isolamento, la cultura e la trasfezione di monociti e macrofagi derivati da monociti. Mentre sono impiegati kit e reagenti disponibili in commercio, il protocollo fornisce importanti suggerimenti e condizioni ottimizzate per il successo dell'aderenza e della trasfezione dei monociti con mimimi e inibitori di miRNA, nonché con i siRNA.

Utilizzando la procedura descritta, sono stati isolati i monociti umani primari di individui affetti da HIV e donatori sani. Tutti i dati qui presentati sono stati ottenuti daHIV: soggetti sottoposti a cART con basso (<20 copie/mL) o carichi virali non rilevabili e normali conteggi di cellule CD4e T. Subito dopo l'isolamento, le cellule sono state macchiate, e la citometria di flusso è stata eseguita per confermare la purezza delle popolazioni di cellule. I risultat...

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

Presentato qui è un protocollo ottimizzato per isolare, coltivare, tradurre e differenziare i monociti primari umani da individui infettati dall'HIV e dai controlli sani.

Isolamento, trasfezione e cultura dei monociti umani primari

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

L'infezione da HIV causa disfunzione immunitaria anche in individui sottoposti a terapia antiretrovirale senza virus rilevabile. Questo metodo consente lo studio della funzione dei monociti che sono regolatori chiave della risposta immunitaria. Abbiamo ottimizzato questa tecnica per l'isolamento, la cultura e la trasfezione dei monociti primari umani.Usiamo questo metodo per comprendere i meccanismi molecolari della disfunzione immunitaria negli individui infetti da HIV, ma può essere applicato a qualsiasi altro studio immunitario che coinvolga i monociti. Se è la prima volta che lavori con il sangue umano, ricorda di seguire i protocolli di biosicurezza appropriati e di trattare tutti i campioni come materiale possibilmente infettivo. Dopo aver raccolto 40 millilitri di sangue intero umano fresco in quattro tubi a vuoto EDTA da 10 millilitri, utilizzare una tecnica sterile per trasferire tutto il sangue in un singolo tubo di propilene conico da 50 millilitri in un armadio di biosicurezza.Seguendo le istruzioni del produttore da un kit di isolamento monocito umano selezionato, aggiungere due millilitri di cocktail di isolamento monocita dal kit al tubo di sangue e vortice le perline magnetiche dal kit per 30 secondi. Aggiungere due millilitri di perline al sangue e utilizzare una pipetta sierologica da 25 millilitri per mescolare accuratamente le perline con il sangue. Dopo cinque minuti a temperatura ambiente, dividere il sangue equamente tra quattro tubi da 50 millilitri e aggiungere 30 millilitri di PBS sterile integrati con un EDTA millimolare ad ogni tubo.Mescolare nuovamente con una pipetta sierologica in plastica da 25 millilitri e posizionare i tubi in supporti magnetici. Dopo 10 minuti, utilizzare una pipetta per disegnare il contenuto dal centro di ogni tubo facendo attenzione a non aspirare più di un millilitro di globuli rossi e distribuire il contenuto del tubo in uno dei quattro nuovi tubi da 50 millilitri. Aggiungere altri 500 microlitri delle perline magnetiche vorticose a ciascun tubo e mescolare delicatamente la soluzione cellulare.Riposizionare i tubi nei supporti magnetici per cinque minuti prima di trasferire attentamente il contenuto dal centro di ciascun tubo in uno dei quattro nuovi tubi da 50 millilitri. Una volta trasferite tutte le sospensioni cellulari, posizionare ogni nuovo tubo da 50 millilitri nei supporti del magnete per cinque minuti prima di trasferire attentamente il contenuto del tubo in un terzo set di quattro nuovi tubi da 50 millilitri. Quindi raccogliere le cellule per centrifugazione e rimosogliere tutti e quattro i pellet cellulari in un totale di 10 millilitri di PBS sterile per il conteggio.Dopo il conteggio, rimescolare i monociti isolati a una volta 10 alla sesta cellule per millilitro di 37 gradi Celsius mezzo RPMI 1640 privo di siero integrato con antibiotici e placcare un millilitro di cellule rimescolate in piastre di 35 millimetri in un armadio di biosicurezza. Quindi posizionare le piastre nell'incubatore di coltura cellulare per 30-60 minuti per consentire alle cellule di aderire ai fondi del pozzo. Per prime per i macrofagi con un fenotipo simile a M1, aggiungere 100 microlitri di siero bovino fetale contenenti 25 nanogrammi per millilitro di GM-CSF a ciascuna piastra.Per innescare macrofagi con un fenotipo simile a M2, aggiungere 100 microlitri di bovini fetali contenenti 50 nanogrammi per millilitro di M-CSF a ciascuna piastra. Per la trasfezione di monociti con imitazioni di microRNA, inibitori o un piccolo RNA interferente, seguendo il protocollo del produttore per il kit di trasfezione, diluire gli RNA selezionati nel buffer a una concentrazione finale di 1,83 micromolare in 10 microlitri di imitazione diluita o inibitore per trasfezione di una per 10 alla quinta quantità di cellule. Per preparare il reagente di trasfezione, aggiungere un microliter del polimero fornito a un tubo di microcentrifugo da 1,5 millilitri e aggiungere immediatamente 90 microlitri del tampone fornito dal kit per ottenere un totale di 91 microlitri di reagente per trasfezione.Vortice il reggente per tre o cinque secondi e pipetta 90 microlitri della soluzione di trasfezione nel tubo contenente 10 microlitri di RNA diluito. Dopo una miscelazione delicata, incubare la soluzione per 15 minuti a temperatura ambiente prima di aggiungere 100 microlitri di complesso di trasfezione a un pozzo di cellule. Dopo quattro ore a 37 gradi Celsius, sostituire il mezzo con tre millilitri di mezzo completo contenente il fattore di crescita appropriato.Il sesto giorno di coltura, sostituire i supernatanti di coltura delle colture M1 con tre millilitri di mezzo integrati con siero bovino fetale, antibiotici, lipopolysaccaride e gamma di interferone e sostituire i supernatanti delle colture M2 con mezzo integrato con siero bovino fetale, antibiotici, M-CSF e interleuchina-4. Dopo 24 ore di coltura, lavare ogni piatto di macrofagi attivati due volte con PBS fresco per lavaggio. Per le analisi dell'RNA e delle proteine, lesinare le cellule attaccate direttamente nelle piastre per consentire la raccolta del contenuto cellulare rilasciato per la loro analisi.Per l'analisi citometrica del flusso, staccare le cellule con due EDTA millimolare in PBS per 10 minuti a 37 gradi Celsius prima di raschiare delicatamente le cellule dai fondi della piastra per consentire la loro raccolta in tubi microcentrifuge da 1,5 millilitri per la loro analisi. Qui vengono mostrati istogrammi rappresentativi di cellule di controllo attivate da M1 e derivate dal paziente con livelli aumentati di cellule attivate da CD80 e CD83 e M2 con livelli aumentati di CD163 e CD209 rispetto alle cellule T0 non attivate. È interessante notare che CD80 e CD83 sembravano essere più espressi nelle cellule derivate dal controllo rispetto alle cellule derivate dall'HIV.La trasfezione di monociti appena raccolti con un microRNA criptato al vicino infrarosso si traduce in un'efficienza di trasfezione superiore al 90% determinata dalla citometria a flusso e dalla microscopia confocale. Inoltre, la trasfezione non riduce significativamente la vitalità delle cellule derivate dal paziente o di controllo indipendentemente dallo stadio di maturazione cellulare o dalle condizioni di trasfezione. La trasfezione si traduce in un'efficace downregolazione del gene bersaglio durante la trasfezione con il piccolo RNA interferente specifico sia al primo giorno che al quarto giorno dopo l'isolamento.Inoltre, le cellule trasfette con imitazione del microRNA dimostrano un aumento da 48 a 72 volte dell'espressione del microRNA rispetto alle cellule non trasfette, mentre tutti i controlli di trasfezione non mostrano cambiamenti apprezzabili. Il numero di cellule placcate è fondamentale per la sopravvivenza. Consigliamo un milione di cellule per piatto da 35 millimetri.La placcatura in mezzo privo di siero migliorerà notevolmente anche l'attacco cellulare. Le cellule ottenute con questo metodo possono essere trattate con citochine o fattori di crescita per studiare risposte differenziali in pazienti di interesse e controlli sani.

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.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

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

Ricerca

Immunologia e infezioni

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.

Generazione di culture Raft organotipiche da cheratinociti umani primari

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.

Isolamento di monociti umani con un doppio centrifugazione in gradiente e la loro differenziazione a macrofagi in coltura cellulare Borse teflonato

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.

Trasfezione altamente efficiente di umani THP-1 da macrofagi 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).

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<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.

Immagini dal vivo di antifungini attività da Human neutrofili e monociti primaria in risposta a<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.

Piccolo RNA trasfezione in cellule primarie Th17 Human Next Generation elettroporazione

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.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

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.

Mouse e umano-derivato cultura dello strato monomolecolare epiteliale gastrica primaria per lo studio della rigenerazione

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 ...