An improved strategy to recover large fragments of functional human neutrophil extracellular traps
Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been impli...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2013-06-01
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| Series: | Frontiers in Immunology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fimmu.2013.00166/full |
| _version_ | 1811243280557408256 |
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| author | Lorena eBarrientos Viviana eMarin-Esteban Luc ede Chaisemartin Luc ede Chaisemartin Vanessa eLievin le moal Catherine eSandre Elsa eBianchini Valerie eNicolas Marc ePallardy Sylvie eChollet-Martin Sylvie eChollet-Martin |
| author_facet | Lorena eBarrientos Viviana eMarin-Esteban Luc ede Chaisemartin Luc ede Chaisemartin Vanessa eLievin le moal Catherine eSandre Elsa eBianchini Valerie eNicolas Marc ePallardy Sylvie eChollet-Martin Sylvie eChollet-Martin |
| author_sort | Lorena eBarrientos |
| collection | DOAJ |
| description | Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme Alu I was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins (histone 3, lactoferrin) were quantified by western blotting, and dsDNA was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase activity and bacterial killing against various strains. Interindividual differences in histone 3, lactoferrin, elastase and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues. |
| first_indexed | 2024-04-12T14:04:41Z |
| format | Article |
| id | doaj.art-d8c2063e67d043eb8e033d76e8570367 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-12T14:04:41Z |
| publishDate | 2013-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-d8c2063e67d043eb8e033d76e85703672022-12-22T03:30:07ZengFrontiers Media S.A.Frontiers in Immunology1664-32242013-06-01410.3389/fimmu.2013.0016652996An improved strategy to recover large fragments of functional human neutrophil extracellular trapsLorena eBarrientos0Viviana eMarin-Esteban1Luc ede Chaisemartin2Luc ede Chaisemartin3Vanessa eLievin le moal4Catherine eSandre5Elsa eBianchini6Valerie eNicolas7Marc ePallardy8Sylvie eChollet-Martin9Sylvie eChollet-Martin10INSERM UMRS 996, Université Paris-SudINSERM UMRS 996, Université Paris-SudINSERM UMRS 996, Université Paris-SudHôpital Bichat, APHPUniversity Paris-SudINSERM UMRS 996, Université Paris-SudUniversity Paris-SudUniversity Paris-SudINSERM UMRS 996, Université Paris-SudINSERM UMRS 996, Université Paris-SudHôpital Bichat, APHPNetosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme Alu I was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins (histone 3, lactoferrin) were quantified by western blotting, and dsDNA was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase activity and bacterial killing against various strains. Interindividual differences in histone 3, lactoferrin, elastase and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.http://journal.frontiersin.org/Journal/10.3389/fimmu.2013.00166/fullNeutrophilcharacterizationneutrophil extracellular trapsquantificationIsolationNEtosis |
| spellingShingle | Lorena eBarrientos Viviana eMarin-Esteban Luc ede Chaisemartin Luc ede Chaisemartin Vanessa eLievin le moal Catherine eSandre Elsa eBianchini Valerie eNicolas Marc ePallardy Sylvie eChollet-Martin Sylvie eChollet-Martin An improved strategy to recover large fragments of functional human neutrophil extracellular traps Frontiers in Immunology Neutrophil characterization neutrophil extracellular traps quantification Isolation NEtosis |
| title | An improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| title_full | An improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| title_fullStr | An improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| title_full_unstemmed | An improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| title_short | An improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| title_sort | improved strategy to recover large fragments of functional human neutrophil extracellular traps |
| topic | Neutrophil characterization neutrophil extracellular traps quantification Isolation NEtosis |
| url | http://journal.frontiersin.org/Journal/10.3389/fimmu.2013.00166/full |
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