CD73 (NT5E, NTE) Antibody Staining Protocol for Immunocytochemistry


Amalia Forte, Dario Siniscalco, and Umberto Galderisi

Second University of Naples, Italy






CD73 (NT5E, ecto-5'-nucleotidase, E5NT, NTE) is a glycosyl phosphatidylinositol (GPI)-anchored purine salvage enzyme expressed on the surface of human T and B lymphocytes. CD73 plays a role in activating T cells. This enzyme is a dimer of 2 identical subunits bound by a glycosyl phosphatidyl inositol linkage to the external face of the plasma membrane. CD73 can be used as a marker of lymphocyte differentiation, as well as a marker to phenotypically characterize mesenchymal stem cells (MSCs).


Cell preparation:


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.




1. Fixation


hMSCs on the chamber were washed three times with PBS, and fixated with 4% paraformaldehyde for 30 min at room temperature (RT).


2. Blocking


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-CD73 (1:250, Cat# ab54217, Abcam, Cambridge, UK).


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.


5. Counterstain:


After being washed three times with PBS, cells were counterstained with bisbenzimide (Hoechst 33258) and mounted with PBS:glycerol (90%).


6. Observation:


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.


7. Results:


Figure: CD73. CD73 immunostaining in human mesenchymal stem cells.