Amalia Forte, Dario Siniscalco, and Umberto Galderisi
Second University of Naples, Italy
Endoglin (CD105), an endothelial homodimeric membrane antigen containing the tripeptide sequence RGD, is expressed on the endothelial cells of capillaries, arterioles and venules, such as on myelocytic leukaemia cells. Endoglin is a glycoprotein and it is part of the transforming growth factor receptor complex that binds several members of the TGFbeta superfamily. Endoglin is also a marker to phenotypically characterize mesenchymal stem cells (MSCs).
Human bone marrow mesenchymal stem cells (hMSCs) were grown in 10% FBS alpha-minimum essential medium (α-MEM), supplied with basic fibroblast growth factor (bFGF) on a glass-slide culture chamber.
hMSCs on the chamber were washed three times with PBS, and fixated with 4% paraformaldehyde for 30 min at room temperature (RT).
After washing 3 times with PBS, non-specific antibody binding was inhibited by incubation for 30 min in blocking solution (1% BSA in PBS).
3. Primary Antibody:
Primary antibodies were diluted in PBS blocking buffer and cells were incubated overnight at 4°C in primary antibodies to mouse anti-endoglin (1:250; Chemicon/Millipore, Billerica, MA).
4. Secondary Antibody and Detection:
Fluorophore-labeled secondary antibodies (1:1000; Molecular Probes/Invitrogen, Paisley, UK) specific to the IgG species used as a primary antibody were used to locate the specific antigens.
After being washed three times with PBS, cells were counterstained with bisbenzimide (Hoechst 33258) and mounted with PBS:glycerol (90%).
Fluorescently labelled cells were viewed with a Zeiss Axioskop (Zeiss, Germany) epifluorescence microscope and the images were captured with a Zeiss Axiocam and Axiovison software.
Figure: CD105. CD105 immunostaining in human mesenchymal stem cells.