Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α.
Nat Protoc
; 7(12): 2103-11, 2012 Dec.
Article
en En
| MEDLINE
| ID: mdl-23154782
ABSTRACT
Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α(+)Sca-1(+) (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ~7 h to complete.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Antígenos Ly
/
Separación Celular
/
Receptor alfa de Factor de Crecimiento Derivado de Plaquetas
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Células Madre Mesenquimatosas
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Citometría de Flujo
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Proteínas de la Membrana
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Nat Protoc
Año:
2012
Tipo del documento:
Article
País de afiliación:
Reino Unido