A method for DNA histogram analysis is described that depends only on the simple assumption that the data are normally distributed and a requirement that a G1 peak is present. A probability density function was derived from the assumption that extracted the S-phase component from the whole histogram. The model was tested with simulated data, and good agreement between predicted and known proportions in G1, S, and G1+M was found. Good agreement was also found between duplicates of experimentally derived data. Some systematic errors are present in the analysis of certain types of histograms. However, these result in small errors when compared with biological and experimental variation and are less than the average of algorithms in current use.The program required only two queued requests, those of the start and the end channels over which the analysis is to be performed. The algorithms perform rapidly on a microcomputer with only 28K addressable memory. Only two failures occurred in over 350 analyses and the method can be used for drug-and radiation-perturbed populations as well as with unperturbed. Key terms: Flow cytometry, DNA histogram analysisA number of models have been proposed for the analysis of flow cytometric DNA histogram data (1, 2, 4-9, 11-18, 25, 28-29) and a comparative review of the various methods has been published (3). .As expected, some models performed better than others, not only with simulated but also with experimentally derived data; however, none was ideal. The models that performed best tended to be those requiring a large mainframe computer, as they generally contained a large number of variables for which a solution had to be found. These include the position of the mean of the G1 and G2+M peaks, the standard deviations of these peaks, the age distribution of the population (22), the rate of DNA synthesis, and the relative durations of the G1, S, and G2 +M phase times with their standard deviations. Thus far, 12 variables have been defined, all of which may have to be considered simultaneously, which is not a trivial task, even with a large computer. Herein lies one of the central problems. The experimental DNA histogram contains, at best, two peaks and a trough corresponding to G1, G2 +M, and S-phase, respectively. This is a very simple data set with which to compute 12 variables, and this must be totally inadequate compared with the complexity of the biology.The work presented in this paper was undertaken to simplify the analysis of DNA histograms and to produce a robust method that gives results rapidly (within 30 s) on a microcomputer with only 28K addressable memory. THEORY AssumptionsIn our attempt to produce a "minimum assumption and computing" model (MAC) we have only assumed that the data are normally distributed. The Computer ProgramThe method of analysis will be illustrated by a description of the program flow diagram and the components of its various steps.Step 1. Eliminate high-frequency noise, if this exists, by "spreading" the data with a constant standard deviation o...

Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the "shape" of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties. Key terms: Hoechst 33342, DNA binding, fluorescence spectrumDuring a series of experiments in which we used Hoechst-33342 (Ho-33342) as a vital DNA stain for mouse bone marrow, we encountered a number of anomalies. These were not only dependent on the dyeDNA concentration ratio and staining time (which were to be expected) but also on the wavelength at which the emission spectrum was analysed. In an attempt to produce an internal biological standard, we repeated the staining procedures with chicken thymocytes (CTHY) and discovered similar anomalies in the "DNA" histograms.The Ho-33342DNA emission spectrum is wide and covers a range from about 400 to 600 nm. Our custombuilt flow cytometer, for which preliminary descriptions have been published (12,13) has the capacity to measure fluorescence emission for up to five different wavelength bands simultaneously. During these experiments we noted that the "DNA" histograms obtained with Ho-33342 staining were considerably different when measured on the violet (390-440 nm) and the green (515-560 nm) photomultipliers. This paper describes our experiences with the chicken thymocytes and discusses some possibilities for the phenomena observed.phate-buf€ered saline, PBS (pH 7.21, the cells were resuspended in Iscove's serum-free medium, pH 7.2, at a concentration of 106/ml. StainingA stock solution of Hoechst-33342 (Calbiochem, La Jolla, CA) was dissolved in distilled water at a concentration of 1 mM. Aliquots of cells were then stained by adding either 1 or 5 p1 of the stock Ho-33342 solution to 1 ml of the cell suspensions to give final stain concentrations of either 1 or 5 pM. The stained cells were then incubated at 37°C in the dark. The samples were analysed at 15, 30, 45, and 125 min after addition of the stain. Flow CytometryThe 164-05 argon laser (Spectra-Physics, Mountainview, CA) of our flow cytometer was tuned to the UV lines (351 + 363 nm), and the fluorescence emission spectrum was analysed on two photomultiplier tubes that admitted light through bandpass filters of 390-440 MATERIALS AND METHODS CellsThymocytes were obtained from a 6-wk-old commercial white-leghorn-based hybrid chicken. A single cell suspension was obtained by teasing the thymus and filtering through ...

Flow cytometry (FCM) has been used to measure intracellular Adriamycin (ADM) in cells cultured in monolayer (Bhuyan et al., 1981, Durand & Olive, 1981, Ganapathi et al., 1982, suspension cultures (Durand & Olive, 1981, Ganapathi et al., 1982, Krishan & Ganapathi, 1980, Sutherland et al., 1979 and in spheroids (Durand, 1981). We have used FCM and cell survival assays to investigate the response of exponentially growing and plateau phase monolayer EMT6 cultures to ADM.A number of problems are associated with FCM measurement of ADM fluorescence (Durand & Olive, 1981)

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Summary.-The reaction rates of enzymes hydrolysing fluorescein diacetate have been studied in populations of intact tissue-culture EMT6, cells using flow cytofluorimetric techniques. It was found that the activity of these enzymes increased in plateau phases and that this correlated inversely with plating efficiency. Highly abnormal substrate-dependent reaction velocity kinetics were found in 14-, 21-, 28-and 35-day cultures. IN previous studies (Watson and Chambers, 1977) it has been shown that an enriched clonogenic fraction of EMT6 cells grown in vivo and separated by their ability to stick to plastic (Twentyman and Watson, 1977) has higher RNA levels than those cells which do not stick to plastic and which have a low plating efficiency. However, as the RNA distributions of the high and low clonogenic fractions overlapped, work was undertaken to find a second biochemical parameter which could be assayed simultaneously with RNA, in order to obtain a better discrimination between the 2 populations. Enzymes hydrolysing fluorescein diacetate can be assayed in populations of intact cells in suspension, using flow cytofluorimetric techniques and it was decided to study these enzymes to see whether hydrolysis rates could be utilized in conjunction with RNA measurements. The work presented in this communication compares the enzyme activities in populations of individual EMT6/M/CC cells during exponential growth and at 5 stages during the plateau phase and the results are related to changes in plating efficiency. MATERIALS AND METHODSEMT6/M/CC cells.-These are a variant of a mouse mammary tumour line (EMT6) (Rockwell, Kallman and Fajardo, 1972) which has been maintained in tissue culture for over 3 years in our laboratories. The preparation of single-cell suspensions and growth kinetic data have been reported previously by Twentyman et al. (1975). For these studies, cells were assayed during exponential growth and during the plateau phase at 7, 14, 21, 28 and 35 days after seeding the monolayer. Immediately before the enzyme assay, the monolayers were trypsinized and the cells were resuspended in medium at a concentration of 1-2 X 106/ml.Five separate experiments were carried out over a number of weeks. At each run, exponentially growing cells were assayed in parallel with one of the 5 ages of plateau phase. The culture medium was changed daily from Day 3 onwards in all plateauphase flasks. Plating efficiencies were also carried out in conjunction with the enzyme studies.Enzyme reaction.-The hydrolysis of fluorescein diacetate (FDA) is catalysed by a variety of hydrolytic enzymes, including lipase, acylase and a-and y-chymotrypsin (Guilbault and Kramer, 1966). We shall refer to these enzymes collectively as esterase. Activity