Isolation and transplantation of allogeneic pulmonary endothelium derived from GFP transgenic mice.
The isolation of primary endothelial cells from murine tissues has long been a challenge and remains a difficult task. Using GFP transgenic C57/BL6 mice as donors, we describe a reliable method to isolate pulmonary endothelial cells by flow cytometry after staining with DiI-Ac-low density lipoprotein (LDL). After mechanical dissociation of murine lung tissue and enzymatic digestion, adherent cells can be quickly stained and sorted by flow cytometry. The isolated cells express endothelial cell markers such as CD31, MECA32 and CD106 and stained positive for Isolectin B4. After 50-fold expansion using standard endothelial growth media, cells could be transplanted into lethally irradiated allogeneic hosts and were detectable using fluorescence microscopy up to 24-h post-transplantation in pulmonary tissue.
Ewing
Patricia
P
Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany.
Wilke
Andrea
A
Brockhoff
Gero
G
Andreesen
Reinhard
R
Eissner
Günther
G
Holler
Ernst
E
Gerbitz
Armin
A
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000637:transplantation*; D005260:Female; D049452:Green Fluorescent Proteins; D008077:Lipoproteins, LDL / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008168:Lung / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D013194:Staining and Labeling; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis
D008077:Lipoproteins, LDL; D008164:Luminescent Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019010:Vascular Cell Adhesion Molecule-1; D049452:Green Fluorescent Proteins