Cell and tissue kinetics最新文献

A flow cytometric live-dead cell assay that uses the dual staining of Hoechst 33342 and propidium iodide (HO-PI) was evaluated for its ability to determine the clonogenicity of treated HeLa cells. The colony-forming assay was used as the reference to determine the capability of the HO-PI assay to measure the proportion of clonogenic cells present in a given population. The viability estimates of both the FCM and trypan blue dye exclusion assays accurately predicted the colony-forming efficiency (CFE) of untreated populations of HeLa cells. However, immediately after treatment with either heat, freeze-thawing, or ionizing radiation the HO-PI assay greatly overestimated the clonogenicity of HeLa cells. This lack of correlation between the FCM determined viability and clonogenic survival was also observed when the cells were heated (45 degrees C, 30 min) and then assayed at 0, 1, 2, 3, 6, 12, 16 or 23 hr after treatment. These data demonstrate that viability as estimated by the HO-PI did not predict survival after acute treatment. In sixty-seven mouse mammary tumour cells, when clonogenicity decreased as a function of time spent in nutrient deprived conditions (chronic treatment), the HO-PI assay again predicted higher CFEs than were measured. Therefore, in these experiments the HO-PI assay could not predict cell death and gave no better measure of cell viability than the trypan blue dye exclusion test.

In this study the metaphase-arrest method with vincristine (VCR) was used, in a transplanted C3H mouse mammary tumour, to study cell proliferation relative to distance from a blood vessel. The metaphase-arrest method has a number of advantages over methods involving [3H]TdR labelling for such a study. The cell birth rate (kB) was shown to be inversely related to distance from a capillary in corded areas of tumour. Penetration of VCR, even into perinecrotic areas of tumour, appeared to be sufficient to achieve effective metaphase arrest.

In vivo effects of colchicine on mitotic counts, DNA synthesis, and cell migration time were investigated in hairless mouse epidermis. [H3]TdR was used to examine DNA synthesis and measure the transit time. We found that colchicine (at a dose of 50 micrograms/25 g body weight) caused mitotic arrest, but had no effect on the number of cells in S phase during the first 4 hr after injection. Colchicine reduced the number of cells in S phase in normal epidermis during the 16-30 hr after injection, but the effect was no longer seen at 42 hr post injection. A similar depression in S-phase cell counts appeared in UVR-stimulated hyperplastic epidermis by 48 hr after injection of colchicine. In addition, migration of [H3]TdR-labelled cells from the basal layer to the superficial layers in UVR-stimulated hyperplastic epidermis was disturbed by colchicine injection. The delay in movement was not seen during the first 24 hr, but appeared by 48 hr after injection.

Using adherent marrow cell layers devoid of stem cells, in vitro cloning and the determination of the frequency of murine haemopoietic stem cells were performed by means of limiting dilution. The presence of stem cells in individual microcultures was indirectly proven by the sustained presence of in vitro colony-forming cells (CFC). These cells increased in number as a function of the culture period, which seems to indicate that the CFC were, de novo, produced in vitro. Although stem cell frequencies comparable to the in vivo spleen colony assay were determined in some experiments, most of our frequency estimates suggested stem cell frequencies ranging from 5/10(6) to 90/10(6). After a period of approximately 4 weeks, the stem cell activity in the microcultures declined rapidly. This may have been caused either by an increased differentiation pressure governed by CSF and/or similar factors or by sub-optimal culture conditions.

Cell cycle parameters, as well as the mode of proliferation of glial cells, in four different areas of the brain of the 14-day-old rat (cortex, corpus callosum, nucleus caudatus putamen and commissura anterior) were studied using different cell kinetic methods after injection of [3H]TdR and/or [14C]TdR. The duration of the S phase (tS) was found to be about 10 hr and that of the cycle time (tC) about 20 hr, tG2 is less than 2 hr and t(G2 + M) about 4 hr. These values are valid for glial cells in all four brain areas studied. However, the labelling index (LI) of the glial cells differs by a factor of 3, between 1.8 and 5.4% in the different brain areas. Accordingly, the growth fraction of the glial cell population in the four areas varies between 0.04 and 0.12. Glial cells (astrocytes as well as oligodendrocytes) proliferate according to a steady state system. Furthermore, the proliferation of glial cells is associated with continuous cell loss. After each mitosis about 3% of the daughter cells become pyknotic and die. In addition, a permanent exchange of glial cells occurs between the proliferating and non-proliferating pool.

The progression of pre-implantation mouse embryos through the first, second and third embryonic cell cycle was investigated cytofluorometrically. In contrast to most of the previous studies, the ova were spontaneously ovulated and the mating period of the ova donors was short (06.00-09.00 hours). The embryonic cells proceeded through the first, second and third cell cycle as a cohort. Thus it was possible to estimate the duration of the cell cycle phases directly from the DNA histograms. The length of the cell cycle phases differed between the embryos of the two different mouse strains. The most pronounced differences were found for the G2 + M phases (first cell cycle: 8 hr for Strain I and 5 hr for Strain II; second cell cycle: 11.5 hr and 14 hr respectively). However, in accordance with previous investigations, common features of the early pre-implantation cell kinetics were also observed: increasing length of the S phases from the first to the second cell cycle and very short G1 phases in the second and third cell cycle. The cell proliferation of the embryos of both strains after the third cell cycle was characterized by exponential growth. The proliferation rate was higher in Strain I embryos than in Strain II embryos (steeper increase of the growth curve). At the end of the pre-implantation development (hatched blastocysts), the growth curves of both strains decreased. The differences concerning the durations of the cell cycle phases and the proliferation rates are considered to be strain specific. It is suggested that the differences in the pre-implantation cell kinetics which have been described generally reflect the strain specificities more than different investigational methods and/or different grades of synchrony of early pre-implantation embryos.

We have studied the pattern of CFU-C disappearance from the peripheral blood of normal and total-body-irradiated dogs given cryopreserved cell suspensions from bone marrow, foetal liver and peripheal blood containing known numbers of CFU-C under an autologous and allogeneic donor-recipient relationship. Only a small fraction of infused donor CFU-C could be detected in the circulation of recipients at the end of the infusion. There was an exponential fall in circulating CFU-C, indicating random loss of infused CFU-C. The CFU-C disappearance pattern in each experimental group was reproducible. The mean half life of autologous blood derived CFU-C in the circulating blood of normal recipients was 8.2 min and the mean blood CFU-C turnover was calculated to be 9.3 X 10(5) CFU-C/kg per day.

In the past, phase fractionation has been used as a determinate of the level of stimulation of cells perturbed by stimulators and inhibitors of DNA synthesis. This method has, in our hands, proven to be insensitive and unreliable with human myeloid cells. A new method of analysis is described in this paper which involves utilization of the magnitude of the slope of a line fit to the mid-portion of S phase as an index of the level of stimulation of cultured human myeloid cells. This method is fast, reliable and simple to implement on an inexpensive microcomputer.

An algorithm is described for the calculation of radioautographic cell grain count from measurements of total cell nuclear area and total grain area. This algorithm provides a statistical correction for grain overlap that is based on the solution to the occupancy problem in probability theory. This method permits the use of automated grain counting over a wide range of grain counts per cell, and extends the useful dynamic range of radioautographic grain counting to well over 200 grains/cell.

A new mathematical approach to the calculation of the kinetics of macrophages in a tissue compartment is presented. This approach, which takes into account the influx of monocytes into the compartment, the local division of mononuclear phagocytes, and the efflux of macrophages from the compartment, was applied to data on the pulmonary macrophages of mice in the normal steady state. The results show that at least 70% of the pulmonary macrophage population is supplied by monocyte influx and at most 30% by local division of immature mononuclear phagocytes originating from the bone marrow. The calculated turnover time of pulmonary macrophages is about 6 days, and the turnover amounts to 14.6 X 10(3) macrophages/hr.