Changes in the surface coat of mesenchymal cells of mouse limb buds after enzymatic cell separation

Isolation of cells is nowadays performed by enzymatic means. The influence of such enzymes on the surface coat of mesenchymal and blastemal cells during the dissociation of limbs buds from 11-day-old mouse embryos was studied electron microscopically after staining with ruthenium red. EGTA or collagenase failed to bring about cell separation. The surface coat seemed to be unchanged after collagenase treatment. After EGTA an increase in extracellular filaments was observed. The proteases alpha-chymotrypsin, dispase II, papain, pronase P and trypsin (0.2%, 37 degrees C, 20 min) succeeded in completely dissociating limb buds. Apart from single granules, there was a detachment of the surface coat from the cells in all cases studied. Hyaluronidase led to only partial separation, but the detachment of the surface coat was almost complete, indicating a GAG-rich surface layer on these cells.