Summarybegin experiments. The usual growth medium was Eaple's mlnimalThirteen cystic fibrosis and 12 normal strains of skin fibroblasts obtained from the Institute for Medical Research were compared for their degree of production of cyclic adenosine 3*:5'-monophosphate in response to isoproterenol and prostaglandin El. There were no significant differences in their quantitative responses in content of cyclic AMP at two different times whether these cells were growing exponentially or were already confluent. AU strains responded similarly to the presence of two types of phosphodiesterase inhibitor. The averaged initial rates of the response to isoproterenol in exponentially growing cells were also similar for the two sets of strains. Although response differed greatly between strains, the response of each strain was relatively reproducible.esGntiaf medium (MEM) plus 1 mM pyruvate, 0 . 2 -m~ asparagine, and 0.2 mM serine (7) and 10 to 20% fetal calf serum. In the experiments of Table 1 we used a medium with 15% fetal calf serum in analogy with Buchwald (1). Cells were freed from the plate for passaging by incubation at room temperature in 0.05% trypsin for 5 to 10 min followed by rigorous pipetting in growth medium. This allowed quantitative release for all strains except GM323. The trypsin was dissolved in an isotonic salts solution (GHCKS) brought to pH 7.4 with sodium hydroxide. Each liter of GHCKS contained 2 g glucose, 4.77 g 4-(2-hydroxyethy1)-1-piperazineethanesulfonic acid (HEPES) (20 mM), 3 g sodium citrate, 0.2 g potassium chloride, 6.37 g sodium chloride, and 0.01 a phenol red. Cells in 100 mm dishes were rinsed once and then -.incubated with 3 ml trypsin solution. In preparation for freezing, Speculation however, cells were incubated in a film of trypsin in a draft-free It appears that cystic fibrosis skin fibroblasts and normal skin area.were then resuspended in 70%M E M j 20% fibroblasts are equally sensitive to isoproterenol. The likely explaserum, and lo% dimethy' sulfoxide and frozen Over nation for a previous observation of a Ifold higher response to liquid nitrogen in a Union Carbide Biological Freezer BF5.isoproterenol is that an improbable selection of the five cystic were grown in 24-we11 plates in fibrosis strains and the five normal strains occurred.1 ml of growth medium at 37°C in 5% COz. In preparation for stimulation of the cells, we set the plates in a 37°C water bath and replaced the growth medium with warmed HEPES-buffered EaThe observation by Buchwald (1) that skin fibroblasts taken gle's MEM (without serum) containing the desired concentration from five boys afflicted with cystic fibrosis (CF) produced more of freshly dissolved agonist. At the appropriate times, this medium cyclic adenosine 3':5'-monophosphate (cyclic AMP) in response was aspirated and replaced with 5% trichloroacetic acid (TCA). to isoproterenol than did strains of fibroblasts taken from normal The TCA supernatants were placed onto Pastew pipette cohmns young males prompted us to test this correlation on larger Sam...

Assays based on the counting of total cells and of colony-forming cells were used to demonstrate that neither dexamethasone nor dibutyryl adenosine 3',5'-monophosphate (cyclic AMP) kills human fibroblasts under a variety of conditions. These results contradict those of previous studies showing that dexamethasone and dibutyryl cyclic AMP kill a higher percentage of fibroblasts from normal humans than from individuals with cystic fibrosis.

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [3H]‐labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [3H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G2, S + G2+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G2, G2+ M and G2+ M + G1. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log‐normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.

No abstract