Abstract
Biology
ERRATUM NOTICE
Important: There has been an erratum issued for this article. Read more …Knoglemarvs megakaryocytter er store polyploidceller, der sikrer produktionen af blodplader. De stammer fra hæmatopoietiske stamceller gennem megakaryopoiesis. De sidste faser af denne proces er komplekse og involverer klassisk de bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) og unipotent Megakaryocyte Progenitors (MKp). Disse populationer går forud for dannelsen af bona fide megakaryocytter, og som sådan kunne deres isolation og karakterisering give mulighed for en robust og upartisk analyse af megakaryocytdannelse. Denne protokol præsenterer i detaljer proceduren for indsamling af hæmatoopoietiske celler fra musebenmarv, berigelse af hæmatoopoietiske forfædre gennem magnetisk udtømning og endelig en cellesorteringsstrategi, der giver stærkt rensede MEP- og MKp-populationer. For det første opsamles knoglemarvsceller fra lårbenet, skinnebenet, og også iliaca crest, en knogle, der indeholder et stort antal hæmatopoietic forfædre. Brugen af iliac crest knogler drastisk øger det samlede celletal opnået pr mus og dermed bidrager til en mere etisk brug af dyr. En magnetisk afstamning udtømning blev optimeret ved hjælp af 450 nm magnetiske perler giver en meget effektiv celle sortering af flow cytometri. Endelig præsenterer protokollen mærknings- og gatingstrategien for sortering af de to stærkt rensede megakarycyt stamfaderpopulationer: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) og MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright ). Denne teknik er let at gennemføre og giver nok cellulært materiale til at udføre i) molekylær karakterisering for en dybere viden om deres identitet og biologi, ii) in vitro differentiering assays, der vil give en bedre forståelse af mekanismerne for modning af megakaryocytter, eller iii) in vitro modeller af interaktion med deres mikromiljø.
Erratum
Erratum: Isolation of Mouse Megakaryocyte ProgenitorsAn erratum was issued for: Isolation of Mouse Megakaryocyte Progenitors. A figure was updated.
Figure 2 was updated from:
Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. Please click here to view a larger version of this figure.
to:
Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. Please click here to view a larger version of this figure.
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