Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro
Summary Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects o...
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| Veröffentlicht in: | Immunology 2020-01, Vol.159 (1), p.63-74 |
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| creator | Nielsen, Marlene C. Andersen, Morten N. Møller, Holger J. |
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Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
Different monocyte‐isolation techniques affect both cell yield, and purity and phenotype of isolated monocytes and monocyte‐derived macrophages in vitro. |
| doi_str_mv | 10.1111/imm.13125 |
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Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
Different monocyte‐isolation techniques affect both cell yield, and purity and phenotype of isolated monocytes and monocyte‐derived macrophages in vitro.</description><identifier>ISSN: 0019-2805</identifier><identifier>EISSN: 1365-2567</identifier><identifier>DOI: 10.1111/imm.13125</identifier><identifier>PMID: 31573680</identifier><language>eng</language><publisher>England: Wiley Subscription Services, Inc</publisher><subject>Adhesion ; Antigens, CD - metabolism ; Antigens, Differentiation, Myelomonocytic - metabolism ; Biomarkers ; Blood ; CD14 antigen ; CD163 ; CD163 antigen ; Cell Adhesion ; Cell culture ; Cell Differentiation ; Cell Separation - methods ; Cells, Cultured ; Flow Cytometry ; Genotype & phenotype ; Histocompatibility antigen HLA ; Humans ; Interleukin-6 - metabolism ; Lectins, C-Type - metabolism ; Leukocytes (granulocytic) ; Leukocytes (mononuclear) ; Lipopolysaccharide Receptors - metabolism ; macrophage ; Macrophages ; Macrophages - immunology ; Macrophages - metabolism ; Macrophages - physiology ; Mannose-Binding Lectins - metabolism ; monocyte ; Monocytes ; Monocytes - immunology ; Monocytes - metabolism ; Monocytes - physiology ; monocyte‐derived macrophage ; Negative selection ; Original ; Peripheral blood mononuclear cells ; Phenotype ; Phenotypes ; Phenotypic plasticity ; Platelets ; Purity ; Receptors, Cell Surface - metabolism ; Tumor Necrosis Factor-alpha - metabolism ; Viability ; Yield</subject><ispartof>Immunology, 2020-01, Vol.159 (1), p.63-74</ispartof><rights>2019 John Wiley & Sons Ltd</rights><rights>2019 John Wiley & Sons Ltd.</rights><rights>Copyright © 2020 John Wiley & Sons Ltd</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4435-a014a31dfccc323780b4945d033039e7b0f9a845f63afd52b79d3ed860bd82693</citedby><cites>FETCH-LOGICAL-c4435-a014a31dfccc323780b4945d033039e7b0f9a845f63afd52b79d3ed860bd82693</cites><orcidid>0000-0003-0820-7789 ; 0000-0003-2008-4224</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904589/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904589/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27903,27904,45553,45554,46387,46811,53769,53771</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31573680$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nielsen, Marlene C.</creatorcontrib><creatorcontrib>Andersen, Morten N.</creatorcontrib><creatorcontrib>Møller, Holger J.</creatorcontrib><title>Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro</title><title>Immunology</title><addtitle>Immunology</addtitle><description>Summary
Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
Different monocyte‐isolation techniques affect both cell yield, and purity and phenotype of isolated monocytes and monocyte‐derived macrophages in vitro.</description><subject>Adhesion</subject><subject>Antigens, CD - metabolism</subject><subject>Antigens, Differentiation, Myelomonocytic - metabolism</subject><subject>Biomarkers</subject><subject>Blood</subject><subject>CD14 antigen</subject><subject>CD163</subject><subject>CD163 antigen</subject><subject>Cell Adhesion</subject><subject>Cell culture</subject><subject>Cell Differentiation</subject><subject>Cell Separation - methods</subject><subject>Cells, Cultured</subject><subject>Flow Cytometry</subject><subject>Genotype & phenotype</subject><subject>Histocompatibility antigen HLA</subject><subject>Humans</subject><subject>Interleukin-6 - metabolism</subject><subject>Lectins, C-Type - metabolism</subject><subject>Leukocytes (granulocytic)</subject><subject>Leukocytes (mononuclear)</subject><subject>Lipopolysaccharide Receptors - metabolism</subject><subject>macrophage</subject><subject>Macrophages</subject><subject>Macrophages - immunology</subject><subject>Macrophages - metabolism</subject><subject>Macrophages - physiology</subject><subject>Mannose-Binding Lectins - metabolism</subject><subject>monocyte</subject><subject>Monocytes</subject><subject>Monocytes - immunology</subject><subject>Monocytes - metabolism</subject><subject>Monocytes - physiology</subject><subject>monocyte‐derived macrophage</subject><subject>Negative selection</subject><subject>Original</subject><subject>Peripheral blood mononuclear cells</subject><subject>Phenotype</subject><subject>Phenotypes</subject><subject>Phenotypic plasticity</subject><subject>Platelets</subject><subject>Purity</subject><subject>Receptors, Cell Surface - metabolism</subject><subject>Tumor Necrosis Factor-alpha - metabolism</subject><subject>Viability</subject><subject>Yield</subject><issn>0019-2805</issn><issn>1365-2567</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU9rFDEYh4NY7Fo9-AUk4EUP0-bvzOQilNJqoYsXPYdMktlJmUnGJLsy4Ic3666lLZhLyJsnD2_eHwDvMDrHZV24aTrHFBP-AqwwrXlFeN28BCuEsKhIi_gpeJ3SfTlSxPkrcEoxb2jdohX4vQ4-6CVb6FIYVXbBw2z14N3PrU0wuY13vdPK53GBbpqVzjAPFs6D9SEvs4Whh13Iw_G9NXA6GhNU3kBjo9vtq0rHMA9qU-rOw53LMbwBJ70ak3173M_Aj5vr71dfq7tvX26vLu8qzRjllUKYKYpNr7WmhDYt6phg3CBKERW26VAvVMt4X1PVG066RhhqTVujzrSkFvQMfD545203WaOtz1GNco5uUnGRQTn59Ma7QW7CTtYCMd7uBR-Pghj2c8lycknbcVTehm2ShAjRNIQzVNAPz9D7sI2-fE-S0jxpGBW8UJ8OVBlKStH2D81gJPeZypKp_JtpYd8_7v6B_BdiAS4OwC832uX_Jnm7Xh-UfwBqUa6A</recordid><startdate>202001</startdate><enddate>202001</enddate><creator>Nielsen, Marlene C.</creator><creator>Andersen, Morten N.</creator><creator>Møller, Holger J.</creator><general>Wiley Subscription Services, Inc</general><general>John Wiley and Sons Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QR</scope><scope>7T5</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-0820-7789</orcidid><orcidid>https://orcid.org/0000-0003-2008-4224</orcidid></search><sort><creationdate>202001</creationdate><title>Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro</title><author>Nielsen, Marlene C. ; Andersen, Morten N. ; Møller, Holger J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4435-a014a31dfccc323780b4945d033039e7b0f9a845f63afd52b79d3ed860bd82693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Adhesion</topic><topic>Antigens, CD - metabolism</topic><topic>Antigens, Differentiation, Myelomonocytic - metabolism</topic><topic>Biomarkers</topic><topic>Blood</topic><topic>CD14 antigen</topic><topic>CD163</topic><topic>CD163 antigen</topic><topic>Cell Adhesion</topic><topic>Cell culture</topic><topic>Cell Differentiation</topic><topic>Cell Separation - methods</topic><topic>Cells, Cultured</topic><topic>Flow Cytometry</topic><topic>Genotype & phenotype</topic><topic>Histocompatibility antigen HLA</topic><topic>Humans</topic><topic>Interleukin-6 - metabolism</topic><topic>Lectins, C-Type - metabolism</topic><topic>Leukocytes (granulocytic)</topic><topic>Leukocytes (mononuclear)</topic><topic>Lipopolysaccharide Receptors - metabolism</topic><topic>macrophage</topic><topic>Macrophages</topic><topic>Macrophages - immunology</topic><topic>Macrophages - metabolism</topic><topic>Macrophages - physiology</topic><topic>Mannose-Binding Lectins - metabolism</topic><topic>monocyte</topic><topic>Monocytes</topic><topic>Monocytes - immunology</topic><topic>Monocytes - metabolism</topic><topic>Monocytes - physiology</topic><topic>monocyte‐derived macrophage</topic><topic>Negative selection</topic><topic>Original</topic><topic>Peripheral blood mononuclear cells</topic><topic>Phenotype</topic><topic>Phenotypes</topic><topic>Phenotypic plasticity</topic><topic>Platelets</topic><topic>Purity</topic><topic>Receptors, Cell Surface - metabolism</topic><topic>Tumor Necrosis Factor-alpha - metabolism</topic><topic>Viability</topic><topic>Yield</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nielsen, Marlene C.</creatorcontrib><creatorcontrib>Andersen, Morten N.</creatorcontrib><creatorcontrib>Møller, Holger J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Chemoreception Abstracts</collection><collection>Immunology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Immunology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nielsen, Marlene C.</au><au>Andersen, Morten N.</au><au>Møller, Holger J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro</atitle><jtitle>Immunology</jtitle><addtitle>Immunology</addtitle><date>2020-01</date><risdate>2020</risdate><volume>159</volume><issue>1</issue><spage>63</spage><epage>74</epage><pages>63-74</pages><issn>0019-2805</issn><eissn>1365-2567</eissn><abstract>Summary
Monocyte‐derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte‐derived macrophages were generated by 5‐day culture with macrophage and granulocyte–macrophage colony‐stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1‐skewed (CD80high HLA‐DRhigh CD163low), whereas negative selection MDMs were M2‐skewed (CD80low HLA‐DRlow CD163high). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.
Different monocyte‐isolation techniques affect both cell yield, and purity and phenotype of isolated monocytes and monocyte‐derived macrophages in vitro.</abstract><cop>England</cop><pub>Wiley Subscription Services, Inc</pub><pmid>31573680</pmid><doi>10.1111/imm.13125</doi><tpages>12</tpages><orcidid>https://orcid.org/0000-0003-0820-7789</orcidid><orcidid>https://orcid.org/0000-0003-2008-4224</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Adhesion Antigens, CD - metabolism Antigens, Differentiation, Myelomonocytic - metabolism Biomarkers Blood CD14 antigen CD163 CD163 antigen Cell Adhesion Cell culture Cell Differentiation Cell Separation - methods Cells, Cultured Flow Cytometry Genotype & phenotype Histocompatibility antigen HLA Humans Interleukin-6 - metabolism Lectins, C-Type - metabolism Leukocytes (granulocytic) Leukocytes (mononuclear) Lipopolysaccharide Receptors - metabolism macrophage Macrophages Macrophages - immunology Macrophages - metabolism Macrophages - physiology Mannose-Binding Lectins - metabolism monocyte Monocytes Monocytes - immunology Monocytes - metabolism Monocytes - physiology monocyte‐derived macrophage Negative selection Original Peripheral blood mononuclear cells Phenotype Phenotypes Phenotypic plasticity Platelets Purity Receptors, Cell Surface - metabolism Tumor Necrosis Factor-alpha - metabolism Viability Yield |
| title | Monocyte isolation techniques significantly impact the phenotype of both isolated monocytes and derived macrophages in vitro |
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