Document Detail

Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α.
MedLine Citation:
PMID:  23154782     Owner:  NLM     Status:  MEDLINE    
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.
Diarmaid D Houlihan; Yo Mabuchi; Satoru Morikawa; Kunimichi Niibe; Daisuke Araki; Sadafumi Suzuki; Hideyuki Okano; Yumi Matsuzaki
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-11-15
Journal Detail:
Title:  Nature protocols     Volume:  7     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-16     Completed Date:  2013-04-16     Revised Date:  2014-02-20    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  2103-11     Citation Subset:  IM    
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MeSH Terms
Antigens, Ly / metabolism*
Cell Culture Techniques
Cell Separation / methods*
Flow Cytometry / methods*
Membrane Proteins / metabolism*
Mesenchymal Stromal Cells / cytology*,  metabolism*
Receptor, Platelet-Derived Growth Factor alpha / metabolism*
Grant Support
G0802350//Medical Research Council; //Medical Research Council
Reg. No./Substance:
0/Antigens, Ly; 0/Ly6a protein, mouse; 0/Membrane Proteins; EC, Platelet-Derived Growth Factor alpha

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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