BISACK ANALYSIS OF THE PHYTOHAEMAGGLUTININ-INDUCED PROLIFERATION OF HUMAN PERIPHERAL LYMPHOCYTES

The rate of stimulation as well as subsequent cell cycle duration was examined in phytohaemagglutinin-stimulated human peripheral lymphocytes grown in vitro in the presence of non-inhibitory concentrations of bromodeoxyuridine. After incorporation of this heavy atom analogue of thymidine into replicating cellular DNA, it was possible to identify unequivocally metaphase cells which had replicated for one, two and three or more cell cycles. Utilizing this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, were analysed by a computer simulation model, and the rate of stimulation (4.5% per hr of the remaining unstimulated population) and cell cycle duration (12·3 hr) were determined. The results were compared with those obtained by autoradiography and the possible relationship to the ‘transition probability’ model for cellular proliferation is discussed.     

Cell cycle kinetics by BrdU-Hoechst flow cytometry: an alternative to the differential metaphase labelling technique

Human lymphocyte cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. The BrdU-Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non-cycling cell fractions and transition probabilities. The Smith and Martin model, modified to include a fraction of non-cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4-6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non-cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. The potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.     

Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

Abstract. The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

Cell Cycle Kinetics By Brdu-Hoechst Flow Cytometry: an Alternative to the Differential Metaphase Labelling Technique

Abstract. Human lymphocytes cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. the BrdU-Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non-cycling cell fractions and transition probabilities. the Smith and Martin model, modified to include a fraction of non-cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4-6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non-cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. the potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.     

Visualization of chromosome assembly during the S and G2 stages of the cycle and chromosome disassembly during the G1 stage in semithin sections of mouse duodenal crypt cells and other cells

Previous examination of dividing cells in the isthmus of the mouse pyloric antrum by using semithin (0.5-micron-thick) Epon sections revealed that the prophasic condensation of chromosomes began early in the DNA-synthesizing (S) stage. In order to examine whether the same observation could be made in other proliferating cell types, the crypt base columnar cells in mouse duodenum and the hepatocytes of the rat 48 hr after partial hepatectomy were investigated by morphologic and radioautographic techniques. When crypt base columnar cells were studied in semithin Epon sections, the four phases of mitosis showed the characteristic features described by classical cytologists. Moreover, the proportion of cells in prophase and telophase was high. To relate the mitotic phases to the stages of the cell cycle, the "frequency of labeled mitoses method" provided the duration of the cell cycle, 12.3 hr, and of the S stage, 7.3 hr. From the frequency of the occurrence of mitotic phases, it was estimated that metaphase lasted 0.3 hr and anaphase 0.11 hr, in line with previous estimates. However, the durations of prophase and telophase were long, 5.9 and 1.9 hr, respectively. The whole mitotic process took over 8 hr. From the duration of prophase and cycle stages, it was calculated that 67% of the S stage was occupied by prophasic cells. In fair agreement with this estimate, 68% of the labeled cells 10 min after a 3H-thymidine injection were found to be in prophase. In regenerating hepatocytes, the morphological features and frequency of prophase and telophase cells were similar to those observed in duodenal crypt cells. While the cycle time was not measured and, therefore, the duration of cycle stages and mitotic phases could not be estimated, it is likely that their duration would be of the same order of magnitude. In conclusion, the mitotic process in duodenal crypt cells takes over 8 hr. Moreover, the crypt cells, like antral isthmal cells, show features of early prophase soon after they enter the S stage of the cycle.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and mice were killed at various intervals after an intravenous injection of 3H-thymidine to time the acquisition of label by the various phases of mitosis. The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G1 and G2 stages were 6.8 and 1.0 hr, respectively. From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurrence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr. Ten minutes after an intravenous injection of 3H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase. We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G2. Also, nuclei are in telophase during the early half of G1 but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G1 into early and late periods is practical.     

Control of cell cycle entry and progression in mitogen-stimulated human B lymphocytes

Tonsillar B lymphocytes were stimulated to proliferate by the mitogenic combination of phorbol dibutyrate and ionomycin. Progression through the cell cycle was monitored by measurements of cellular DNA and RNA content using flow cytometry. Changes in surface expression of class II MHC antigens and CD20 antigen were also monitored as early parameters of B lymphocyte activation and cell cycle progression. The results showed that about 60% of the population synchronously entered and progressed through the cell cycle. The transition from the resting state, signaled by increased RNA content, occurred about 12 to 24 hr after stimulation; S phase entry occurred at about 36 hr. Small, variable populations of cells appeared to be unresponsive to the stimuli, either because they were “preactivated” before in vitro stimulation or were already dying. The kinetics of appearance and accumulation of several cell cycle regulated/regulatory proteins were followed by immunoblotting. The proliferating cell nuclear antigen (PCNA) cyclin A and p33^cdk2 proteins were either absent or present in very low amounts in resting cells and first became detectable in increased amount beginning at about 24 hr after stimulation; increased p34^cdc2 protein was not detected until about 36 hr. Increased cellular content and phosphorylation of the p110^Rb protein was already obvious by 24 hr after stimulation. The effects of several immunosuppressive agents were examined using purified B cells. Both cyclosporin A and an FK506 analogue were shown to inhibit proliferation of B lymphocytes, at the low doses also inhibitory to T cells. © 1995 Wiley-Liss, Inc.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Cytokinesis is more rapid in Ha-T24-ras transfected rat embryo fibroblasts than in non-transfected control cells.

It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that mitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha-T24-ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alterations induced by transfection with Ha-T24-ras accelerate microfilament-dependent cytokinetic furrowing without significant effects on microtubule-dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.     

Time-dependent effects of removal of the pars distalis of the pituitary gland on toad epidermal cell and tissue kinetic parameters

Abstract. Amphibian moulting and its hormonal control has been extensively studied, but the possible influence of hormones on epidermal proliferation has been less investigated. In the present contribution to studies on the control of epidermal homeostasis, the proliferative pattern of the toad epidermis following ablation of the pars distalis of the pituitary gland is analysed by an investigation of the changes in the epidermal cell number, metaphase index and [³H]thymidine incorporation at various times after the operation. During the first 24 hr after pars distalis ablation, labelling index (LI) increased concurrently with an initial decrease in the metaphase index (MI), followed by an increase. During the same period of time the mean grain count (MGC) and the grain distribution pattern also changed. From 24 hr to 7 days after operation, MI, LI, and MGC were significantly lower than those of controls, but increased to control levels at 14 days after the operation. Phase durations and their possible changes were not measured directly, but data showed that the S-phase duration (Ts) and the mitotic duration (TM) must have changed in relation to each other during the experiment. Assuming that the MGC is a rough estimate of the DNA-synthesis rate, the compatibility of a postulated change in phase duration with the observed MGC was analysed. This analysis revealed that TS and TM could have decreased up to 18 hr after the operation, whereas these phase durations, after 24 hr and during the rest of the experiment, increased compared to those of controls. Even with these possible changes in phase durations, and in the absence of direct assessment of cell division rates, the observed cell kinetic parameters appeared incompatible with an increased rate of proliferation. This was surprising since the efflux of cells from the living, subcorneal epidermis to the stratum corneum was significantly increased after pars distalis ablation, without a concurrent decrease in the stratum corneum recruitment cell pool (SCRP-number of subcorneal epidermal cells per mm surface). Possible reasons for the discrepancy between the expected increase in proliferation following pars distalis ablation, and the failure to demonstrate this, are discussed.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

Epidermal tissue homeostasis. I. Cell pool size, cell birth rate and cell loss by moulting in the intact toad, Bufo bufo

Toad epidermis is a suitable model for studies on tissue homeostasis because cell pool size, influx into and efflux from the cell pool can be easily determined. The cell pool size was obtained by cell counting on photomicrographs, the influx (cell birth rate) was assessed by the metaphase-arrest technique, and the efflux (cell loss by moulting) assessed by counting the number of cells in the corneal layer and recording of intermoult periods. The importance of the methods for assessing these parameters per square unit of skin surface is emphasized. These parameters were studied in eight groups of ten adult male toads sacrificed at various hours of the day. There were minor variations in the cell birth rate, fluctuating around a mean of 26 cells/mm2/hr (obtained at the metaphase collection period from 11.00-14.00 hours). By summation of the cell productions during the eight metaphase collection periods of 3 hr, and extrapolation to an intermoult period (time between two moults), a calculated cell production of about 6340 cells/mm2 in 10.3 days was obtained, whereas the cell loss at each moult was only 2370 cells/mm2. Thus the cell production rate exceeds the rate of cell loss through moults by a factor of 2.7 Arguments are presented that the 'surplus' of cells produced cannot be permanently accommodated within the living epidermis. Consequently a cell deletion rate beyond that by moulting of about 4000 cells/mm2 in 10.3 days or 16 cells/mm2/hr can be calculated. These results are discussed in relation to current concepts of tissue homeostatic mechanism(s). The results are consistent with the hypothesis that controlled cell deletion may be a tissue homeostatic mechanism complementary to controlled cell divisions.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Abstract. Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G₂+ M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram.
The cohort can be described as the recruitment of cells from a pre-existing G₀ compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G₂+ M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C

Accessory cell-depleted T cells required the presence of a protein kinase C (PKC) stimulating phorbol ester, such as phorbol 12,13-dibutyrate (PDB), to be activated by soluble antibodies to the CD3 molecular complex. To determine the duration of PDB costimulation necessary to induce a proliferative response, highly purified T cells were pulsed with anti-CD3, incubated with PDB for limited periods of time, and then washed and recultured in the absence of PDB. T cells stimulated with anti-CD3 and PDB for 2 hr were unable to proliferate unless IL-2 or PDB was added to the second culture. With more prolonged exposure to PDB (4-18 hr), anti-CD3-pulsed cells exhibited an increased capacity to proliferate in the absence of additional PDB. Proliferation could be augmented by exogenous IL-2, but remained submaximal. Optimal DNA synthetic responses required the presence of PDB throughout the entire culture. Despite this, costimulation with anti-CD3 and PDB induced a significant number of cells to express IL-2 receptors and enter the cell cycle after 18 hr of costimulation with PDB. Moreover, T cells costimulated by anti-CD3 and PDB produced IL-2 within 4 hr. However, T cells that were stimulated with anti-CD3 and PDB for 4 hr, washed, and recultured rapidly lost the ability to continue to produce IL-2, which reflected a decrease in the content of mRNA encoding IL-2. This loss of IL-2 production was prevented by reculturing the cells with PDB. These studies therefore indicate that after initial T cell activation by anti-CD3, continued stimulation of PKC is necessary for ongoing IL-2 production. These results suggest a model of T cell activation in which sustained stimulation of PKC after cell cycle entry is required to maintain growth factor production and continued proliferation.     

The effects of varying concentrations of colchicine on the progression of grasshopper neuroblasts into metaphase

The effects of four concentrations of colchicine (2.5 x 10^-7, x 10^-5, x 10^-3, and x 10^-2 M) on the cell cycle of grasshopper neuroblasts have been determined by direct observations on living cells. The lowest concentration, 2.5 x 10^-7 M, does not completely disorganize the spindle but does retard its action. The three higher concentrations disorganize the spindle, so that all cells reaching metaphase are blocked in a c-mitotic condition throughout the period of observations (308 min at 38°C, the minimum duration of the cell cycle in untreated neuroblasts). Continuous treatment with all concentrations reduces the rate at which neuroblasts enter metaphase, the extent of the reduction being a function of increasing concentration and time of exposure. After a short exposure to 2.5 x 10^-5 M colchicine, the neuroblasts recover from the inhibiting effects on progression through the cycle to metaphase, but they show no recovery from the inhibiting effects on spindle formation for more than 3 hr. Apparent stimulation of progression rate occurs early in exposure to all concentrations and during recovery from a short exposure to 2.5 x 10^-5 M. Morphological alterations in the chromatin of telophase, interphase, and prophase cells are induced by the higher concentrations of colchicine. The data indicate that caution should be exercised in the use of colchicine for determining cell cycle duration and/or the effects of physical and chemical agents on the cycle.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.