We used a
bacterial neutral protease to disperse KHT solid
tumors into single cell
suspensions suitable for routine cell kinetic analysis by flow cytometry and for clonogenic cell survival. Neutral
protease disaggregation under conditions which would be suitable for routine
tumor dispersal was compared with a
trypsin/
DNase procedure. Cell yield, clonogenic cell survival,
DNA distributions of untreated and
drug-perturbed
tumors, rates of radioactive precursor incorporation during the cell cycle, and preferential cell cycle phase-specific cell loss were investigated.
Tumors dispersed with neutral
protease yielded approximately four times more cells than those dispersed with
trypsin/
DNase and approximately a 1.5-fold higher plating efficiency in a semisolid
agar system. Quantitative analysis of
DNA distributions obtained from untreated and
cytosine-arabinoside-perturbed
tumors produced similar results with both dispersal procedures. The rates of incorporation of tritiated
thymidine during the cell cycle were also similar with neutral
protease and
trypsin/
DNase dispersal. Preferential phase-specific cell loss was not observed with either technique. We find that neutral
protease provides good single cell
suspensions of the KHT
tumor for cell survival measurements and for cell kinetic analysis of
drug-induced perturbations by flow cytometry. In addition, the high cell yields facilitate electronic cell sorting where large numbers of cells are often required.