Pronase is a proteolytic enzyme recently introduced as an effective dispersing agent of tissue culture cells. We have found it to be a most rapid and complete dispersing agent for primary fibroblastic cell lines and clearly superior to trypsin. O n the other hand, with certain continuous epithelial cell lines its completeness of dispersion is inferior to that of trypsin necessitating study of each cell line individually with both enzymes to select the most advantageous one.Since Gwatkin and Thompson ('64) pointed out advantages of dispersing cells in vitro with the crude proteolytic enzyme preparation, pronase, considerable differences have been expressed on its value. Kahn, Ashwood-Smith and Robinson ('65) have noted failure of their cell lines to survive following dispersion with pronase. On the other hand, Sullivan and Schafer ('66) have used the enzyme over approximately two years without difficulty and feel it has been the single most satisfactory enzyme used in their laboratory for producing single cell suspensions without clumps. Weinstein ('66) has confirmed Gwatkin and Thompson's original observation finding that the enzyme more rapidly disperses diploid fibroblastic cells and more effectively eliminates clumping than trypsin. He also showed that there was no significant effect on the number of cell divisions of a diploid fibroblastic strain nor on the chromosome morphology.Our five year's experience with pronase as a cell dispersing agent is contained in the ensuing manuscript. MATERIALS AND METHODSCell lines. Continuous: HeLa cell line 299 and KB cells (obtained from Microbiological Associates). Monkey kidney epithelium was obtained from Dr. George Dubes of the Department of Microbiology.The fibroblastic cell lines were grown from aseptically collected surgical specimens which were minced into 1 mm cubes and placed in growth medium. Primary.J. CELL. PHYSIOL., 75: 159-162. M e d i u m .HeLa cells were grown in a modified Eagle's basal medium (Treadwell and Ross, '62) containing 10% human serum and 10% fetal calf serum.KBM cells were grown in the above medium with 10% calf serum.The primary cultures were grown in a modified medium 199 (Treadwell and Ross, '62) containing 10% human serum and 10% fetal calf serum.Enzymes. Trypsin (Nutritional Biochemical Company, 1 : 300) was made up as a 2% solution in sterile balanced salt solution and stored in aliquots at -20" C.Pronase (Cal. Biochem. Co. B-Grade) was made up in a balanced salt solution at 1% concentration and stored at -20" C until used. Once thawed the enzyme is stored at 4" C and used over a one to two week period without loss of noticeable dispersing potency. Subculture m e t h o d sThe continuous cultures were routinely passed by draining growth medium and adding a balanced salt solution containing 0.2% trypsin for 20 minutes at 36.5" C . Then the cells were gently dispersed by pipetting six times followed by centrifugation at 60 X g for six minutes. The cell pellet was then redispersed in growth medium after discarding the supernate. The ...

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The effects of several concentrations of NO2, NaNO3, and NaNO2 respectively, upon strain L, mouse liver cells, and HeLa cells, were studied and a modified system designed to permit continuous exposure of cells to air pollutants is described. In NCTC medium 109 containing serum, cells tolerate concentrations of NO2 up to 4100 p.p.m. and some may even tolerate 8600 p.p.m. Removal of the serum lowers the lethal concentration of NO2 to less than 100 p.p.m. If the cells were covered only by a thin film of BSS (balanced salt solution) medium, a concentration of 100 p.p.m. NO2 proved toxic within [Formula: see text] hour. If, however, the NO2 concentration was reduced to 5 or 10 p.p.m., cells survived a daily 8-hour exposure but many, if not most, of the cells were dead after several days. The presence of as little as 25 mg% NaNO2 retarded proliferation. On the other hand, NaNO3 was tolerated well in the three cell lines tested; HeLa cells seemed to be the most sensitive of the cell strains with respect to these salts.

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