Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration
Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isola...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2023-12-01
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| Series: | Platelets |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/09537104.2023.2192289 |
| _version_ | 1797684094803378176 |
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| author | Markus Spindler Kristina Mott Harald Schulze Markus Bender |
| author_facet | Markus Spindler Kristina Mott Harald Schulze Markus Bender |
| author_sort | Markus Spindler |
| collection | DOAJ |
| description | Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70–80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like $${\rm{\beta }}$$1-tubulin, $${\rm{\beta }}$$3-integrin, GPVI and GPIb$${\rm{\alpha }}$$, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods. |
| first_indexed | 2024-03-12T00:25:33Z |
| format | Article |
| id | doaj.art-c82f2bb70f834c359ca2a9514d710846 |
| institution | Directory Open Access Journal |
| issn | 0953-7104 1369-1635 |
| language | English |
| last_indexed | 2024-03-12T00:25:33Z |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Platelets |
| spelling | doaj.art-c82f2bb70f834c359ca2a9514d7108462023-09-15T10:38:11ZengTaylor & Francis GroupPlatelets0953-71041369-16352023-12-0134110.1080/09537104.2023.21922892192289Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtrationMarkus Spindler0Kristina Mott1Harald Schulze2Markus Bender3University Hospital WürzburgUniversity Hospital WürzburgUniversity Hospital WürzburgUniversity Hospital WürzburgMegakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70–80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like $${\rm{\beta }}$$1-tubulin, $${\rm{\beta }}$$3-integrin, GPVI and GPIb$${\rm{\alpha }}$$, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.http://dx.doi.org/10.1080/09537104.2023.2192289bone marrowmegakaryocyte enrichmentmegakaryocyte isolationmurineprimary,protocol |
| spellingShingle | Markus Spindler Kristina Mott Harald Schulze Markus Bender Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration Platelets bone marrow megakaryocyte enrichment megakaryocyte isolation murine primary, protocol |
| title | Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| title_full | Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| title_fullStr | Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| title_full_unstemmed | Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| title_short | Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| title_sort | rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration |
| topic | bone marrow megakaryocyte enrichment megakaryocyte isolation murine primary, protocol |
| url | http://dx.doi.org/10.1080/09537104.2023.2192289 |
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