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

Авторы хотели бы поблагодарить ВИЧ клинической / опухолевой биопозитория core для предоставления пациентов образцов и клеточной иммунологии Метаболизм ядро для обеспечения потока цитометрии анализа. Этот проект был профинансирован NIH P20GM12288 и P30GM114732.

Все методы, описанные ниже, были одобрены Университетом штата Луизиана Центр медицинских наук Нью-Орлеан Институциональный обзор совета. Вся кровь была собрана после получения информированного согласия.
ПРИМЕЧАНИЕ: Вся процедура выполняется в стерильных условиях в объ...

<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. 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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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Regulation+of+Macrophages+Polarization+by+MicroRNAs.">Transcriptional Regulation of Macrophages Polarization by MicroRNAs.</a> Frontiers in Immunology. 9, 1175 (2018).</li><li>Hu, X., 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=Genome-Wide+Analyses+of+MicroRNA+Profiling+in+Interleukin-27+Treated+Monocyte-Derived+Human+Dendritic+Cells+Using+Deep+Sequencing:+A+Pilot+Study.">Genome-Wide Analyses of MicroRNA Profiling in Interleukin-27 Treated Monocyte-Derived Human Dendritic Cells Using Deep Sequencing: A Pilot Study.</a> International Journal of Molecular Sciences. 18 (5), (2017).</li><li>Huang, 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=MicroRNA+miR-126-5p+Enhances+the+Inflammatory+Responses+of+Monocytes+to+Lipopolysaccharide+Stimulation+by+Suppressing+Cylindromatosis+in+Chronic+HIV-1+Infection.">MicroRNA miR-126-5p Enhances the Inflammatory Responses of Monocytes to Lipopolysaccharide Stimulation by Suppressing Cylindromatosis in Chronic HIV-1 Infection.</a> Journal of Virology. 91 (10), (2017).</li><li>Lodge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+MicroRNAs-221+and+-222+Inhibit+HIV-1+Entry+in+Macrophages+by+Targeting+the+CD4+Viral+Receptor.">Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor.</a> Cell Reports. 21 (1), 141-153 (2017).</li><li>Ma, L., Shen, C. 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. 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Представленный протокол демонстрирует использование первичных клеток ВИЧ-инфицированных испытуемых в качестве модели для изучения моноцитов и макрофагов. ВИЧ-и пациенты, проходящие ЛЕЧЕНИЕ, живут с инфекцией в течение нескольких лет, а также могут иметь другие сопутствующие ин...

Инфекция вируса иммунодефицита человека-1 (ВИЧ-1) вызывает тяжелую иммунную дисфункцию, которая может привести к оппортунистическим инфекциям и приобретенному синдрому иммунодефицита (СПИД). Хотя ВИЧ-инфицированных пациентов, проходящих CART характеризуются низкими вирусными нагрузками и нормальной CD4И Т-клеток рассчитывает, функционирование иммунной системы может быть поставлена под угрозу в этих людей, что приводит к дисфункциональной иммунной реакции, которая была связана с повышенным риском развития рака1. Механизмы иммунной дисфункции у ВИЧ-инфицированных пациентов на КАРТ остаются в значительной степени неизвестными. Поэтому характеристика иммунных клеток, полученных пациентом, и изучение их биологии и функции является важнейшим компонентом текущих исследований в области ВИЧ.
Моноциты и макрофаги являются ключевыми регуляторами иммунных реакций и играют основополагающую роль в ВИЧ-инфекции2,3,4,5. Неоднородные и пластиковые в природе, макрофаги могут быть широко классифицированы в классическо активированных (M1) или альтернативно активированных (M2). Хотя эта общая классификация необходима при создании экспериментальных условий, статус поляризации макрофагов может быть обращен вспять различными цитокинов6,7,8,9. Хотя несколько исследований исследовали влияние ВИЧ-инфекции на моноциты и дендритные клетки, молекулярные детали моноцитных реакций в значительной степени неизвестны6,7,10,11,12,13,14,15,16,17,18,19. Среди факторов, участвующих в регуляции и функции иммунных клеток, микроРНК (миРНК), короткие некодировочные РНК, которые после транскрипционно регулируют экспрессию генов, были показаны, чтобы играть важную роль в контексте основных клеточных путей (т.е. рост, дифференциация, развитие, и апоптоз)20. Эти молекулы были описаны как важные регуляторы транскрипционных факторов, необходимых для диктовки функциональной поляризации макрофагов21. Исследована потенциальная роль миРНК в моноцитах ВИЧ-инфицированных лиц, проходящих ЦАРТ, но прогресс в этой области требует гораздо больше работы22,23,24,25,26. В настоящем документе обсуждается оптимизированный метод перевода миРНК и сирны в первичные моноциты человека от ВИЧ-инфицированных пациентов и контроля.
Этот протокол опирается на коммерчески доступные реагенты и комплекты, так как непрерывность технической процедуры помогает устранить ненужные экспериментальные переменные при работе с клиническими образцами. Тем не менее, метод предоставляет важные советы (т.е. количество клеток, покрытых или краткое инкубации с сыворотки свободной среды для содействия присоединению клеток к пластине). Кроме того, условия поляризации, используемые в этом протоколе, вытекают из опубликованной работы27,28,29.

<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

Вирус иммунодефицита человека (ВИЧ) остается серьезной проблемой для здоровья, несмотря на внедрение комбинированной антиретровирусной терапии (САРТ) в середине 1990-х годов. В то время как антиретровирусная терапия эффективно снижает системную вирусную нагрузку и восстанавливает нормальные количество CD4и Т-клеток, она не восстанавливает полностью функциональную иммунную систему. Дисфункциональная иммунная система у ВИЧ-инфицированных лиц, проходящих КРТ, может характеризоваться иммунной активацией, ранним старением иммунных клеток или постоянным воспалением. Эти условия, наряду с сопутствующими факторами, связанными с ВИЧ-инфекцией, усугубляют сложность болезни, которая не может быть легко воспроизведена в клеточных и животных моделях. Для исследования молекулярных событий, лежащих в основе иммунной дисфункции у этих пациентов, система культуры и манипулировать человеческими первичными моноцитами in vitro представлена здесь. В частности, протокол допускает культуру и трансфекцию первичных монцитов CD14и, полученных от ВИЧ-инфицированных лиц, проходящих ЦАРТ, а также вич-отрицательных мер контроля. Метод включает в себя изоляцию, культуру и трансфекцию моноцитов и моноцитов, полученных макрофагами. В то время как коммерчески доступные комплекты и реагенты используются, протокол обеспечивает важные советы и оптимизированные условия для успешного присоединения и трансфекции моноцитов с миРНК мимикрирующими и ингибиторами, а также с siRNAs.

Используя описанную процедуру, были выделены первичные моноциты человека от ВИЧ-инфицированных людей и здоровых доноров. Все данные, представленные здесь, были получены из ВИЧ-испытуемых, подвергаясь АРТ с низким (злт;20 коп/мл) или необнаруживаемыми вирусными на...

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

Здесь представлен оптимизированный протокол для изоляции, культивирования, трансфектирования и дифференциации первичных моноцитов человека от ВИЧ-инфицированных лиц и здорового контроля.

Изоляция, Трансфекция и культура первичных человеческих моноцитов

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

ВИЧ-инфекция вызывает иммунную дисфункцию даже у лиц, проходящих антиретровирусную терапию без обнаруженного вируса. Этот метод позволяет изучать функции моноцитов, которые являются ключевыми регуляторами иммунного ответа. Мы оптимизировали этот метод для изоляции, культуры и трансфекции первичных моноцитов человека.Мы используем этот метод, чтобы понять молекулярные механизмы иммунной дисфункции у ВИЧ-инфицированных людей, но он может быть применен к любым другим иммунным исследованиям с участием моноцитов. Если вы впервые работаете с человеческой кровью, не забудьте следовать надлежащим протоколам биобезопасности и рассматривать все образцы как возможно инфекционный материал. После сбора 40 миллилитров свежей человеческой цельной крови в четырех 10 миллилитров EDTA вакуумных трубок, использовать стерильную технику для передачи всей крови в один 50 миллилитров конического пропилена трубки в биобезопасности кабинета.Следуя инструкциям производителя из выбранного человека комплект изоляции моноцитов, добавить два миллилитров моноцитов изоляции коктейль из комплекта в трубку крови и вихрь магнитных бусин из комплекта в течение 30 секунд. Добавьте в кровь два миллилитров бисера и используйте серологическую пипету на 25 миллилитров, чтобы тщательно смешать бисер с кровью. После пяти минут при комнатной температуре, разделить кровь поровну между четырьмя 50 миллилитров трубки и добавить 30 миллилитров стерильных PBS дополняется одним миллимолярной ЭДТА для каждой трубки.Снова смешайте с пластиковой 25 миллилитров серологической пипетки и поместите трубки в магнитных держателей. Через 10 минут используйте пипетку, чтобы составить содержимое из центра каждой трубки, заботясь, чтобы не аспирировать более одного миллилитра красных кровяных телец и распределить содержимое трубки в одну из четырех новых 50 миллилитров труб. Добавьте 500 дополнительных микролитров вихревых магнитных бусин к каждой трубке и аккуратно смешайте клеточный раствор.Поместите трубки обратно в магнитные держатели в течение пяти минут, прежде чем тщательно передавать содержимое из центра каждой трубки в одну из четырех новых 50 миллилитров труб. Когда все подвески клетки были перенесены, поместите каждую новую 50-миллилитровую трубку в магнитные держатели в течение пяти минут, прежде чем тщательно перенести содержимое трубки в третий набор из четырех новых 50 миллилитров труб. Затем соберите клетки путем центрифугации и повторно посовечите все четыре клеточные гранулы в общей сложности 10 миллилитров стерильных PBS для подсчета.После подсчета, resuspend изолированных моноцитов в один раз от 10 до шестой клетки на миллилитр 37 градусов по Цельсию сыворотки свободной RPMI 1640 среды дополняется антибиотиками и пластины один миллилитр resuspended клеток в 35 миллиметров пластин в биобезопасности кабинета. Затем поместите пластины в инкубатор культуры клеток на 30-60 минут, чтобы клетки придерживались дна. Чтобы загрунтовывать макрофаги с фенотипом M1, добавьте 100 микролитров сыворотки крупного рогатого скота плода, содержащей 25 нанограмм на миллилитр ГМ-CSF к каждой пластине.Чтобы загрунтовывать макрофаги с фенотипом M2, добавьте 100 микролитров крупного рогатого скота плода, содержащих 50 нанограмм на миллилитр M-CSF к каждой пластине. Для моноцитов трансфекции с микроРНК имитирует, ингибиторы, или небольшой вмешательства РНК, следуя протоколу производителя для трансфекции комплект, разбавить выбранных РНК в буфере до конечной концентрации 1,83 микромолара в 10 микролитров разбавленной имитации или ингибитора на трансфекцию один раз от 10 до пятого объема клеток. Чтобы подготовить трансфектальный реагент, добавьте один микролитр предоставленного полимера в 1,5 миллилитровую микроцентрифуговую трубку и немедленно добавьте 90 микролитров буфера, предоставленного комплектом, чтобы получить в общей сложности 91 микролитр реагента на трансфекцию.Вихрь регента в течение трех-пяти секунд и пипетка 90 микролитров трансфектного раствора в трубку, содержащую 10 микролитров разбавленной РНК. После нежного смешивания инкубировать раствор в течение 15 минут при комнатной температуре, прежде чем добавить 100 микролитров трансфектного комплекса в одну колодец клеток. После четырех часов при 37 градусах по Цельсию замените среду тремя миллилитровами полной среды, содержащими соответствующий фактор роста.На шестой день культуры замените культурные супернатанты из культур М1 тремя миллилитровами среды, дополненными сывороткой крупного рогатого скота плода, антибиотиками, липополисахаридом и интерфероном гамма и замените супернатанты из культур М2 средой, дополненной сывороткой из крупного рогатого скота плода, антибиотиками, M-CSF и интерлейкином-4. После 24 часов культуры, мыть каждую тарелку активированных макрофагов два раза со свежим PBS за стирку. Для анализа РНК и белка, подлизать прилагаемые клетки непосредственно в пластинах, чтобы сбор выпущенного содержимого клеток для их анализа.Для цитометрического анализа потока, отсоедините клетки с двумя миллимолярной ЭДТА в PBS в течение 10 минут при 37 градусах по Цельсию, прежде чем аккуратно соскоб клетки от дна пластины, чтобы их сбор в 1,5 миллилитров микроцентрифуг труб для их анализа. Здесь показаны репрезентативные гистограммы M1-активированного контроля и клеток, полученных пациентом, с повышенным уровнем CD80 и CD83 и M2-активированных клеток с повышенным уровнем CD163 и CD209 по сравнению с неактивными Т0-клетками. Интересно, что CD80 и CD83, как представляется, более высоко выражены в клетках, полученных из-под контроля, по сравнению с клетками, полученными из ВИЧ.Трансфекция свежесобранных моноцитов с скремблированной почти инфракрасной помеченной микроРНК приводит к более чем 90%-ной эффективности трансфекции, определяемой цитометрией потока и конфокарной микроскопией. Кроме того, трансфекция не значительно снижает жизнеспособность клеток, полученных пациентом, или контрольных клеток, независимо от стадии созревания клеток или условий трансфекции. Трансфекция приводит к эффективному downregulation целевого гена при трансфекции с конкретными малых вмешательства РНК в один день и день четыре после изоляции.Кроме того, клетки, трансфицированные микроРНК-мимикой, демонстрируют 48-72-кратное увеличение экспрессии микроРНК по нетрансфицированным клеткам, в то время как все элементы управления трансфектом не показывают заметных изменений. Количество потроха клеток имеет решающее значение для выживания. Рекомендуем один миллион клеток на 35-миллиметровую тарелку.Покрытие в среде, свободной от сыворотки, также значительно улучшит клеточное вложение. Клетки, полученные с помощью этого метода можно лечить с цитокинами или факторами роста для изучения дифференциальной реакции у пациентов, представляющих интерес и здорового контроля.

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.

Человеческий Т-лимфоцитов изоляции, культуры и анализ миграции 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 ...

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.

Генерация органотипической культур Плот из первичных кератиноцитов человека

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.

Выделение моноцитов человека, дважды градиентного центрифугирования и их дифференциации с макрофагами в тефлоновым покрытием культуре клеток Сумки

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.

Высокоэффективная Трансфекцию человека THP-1 макрофагов по 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).

Выделение и внутривенное введение мышиных костного мозга моноцитов

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.

Лимфоцит Изоляция из человеческой кожи для фенотипического анализа и<em&gt; Ex Vivo</em&gt; Культура клеток

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.

Живая съемка противогрибковой активности на человеке первичных нейтрофилов и моноцитах в ответ на<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.

Малые РНК Трансфекция в первичных человеческих Th17 клеток с помощью следующего поколения электропорации

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.

Изоляции и в пробирке культуры мышиных и человека альвеолярные макрофаги

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.

Мыши и человека, полученных первичных желудка эпителиальных монослойном культивировании для изучения регенерации

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 ...