Simple method for culture of peripheral blood lymphocytes of Testudinidae

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

Fully automated TV-image analysis of the cell-cycle: comparison of the PLM method with determinations of the percentage and the DNA content of labelled cells

A cell-cycle analysis based on a fully automated TV-image scanning system is proposed to replace the laborious PLM method. To compare the efficiency of the two procedures, cell-cycle parameters were assessed in Ehrlich (diploid and hyperdiploid), L-1210, and JB-1 mouse ascites tumours and in rat jejunal crypts. The percentages of labelled mitoses (PLM) were counted visually on Feulgen-stained autoradiographs obtained at various times after a single 3H-thymidine pulse. The fraction of labelled cells (P) and the DNA ratio of labelled and unlabelled cells were measured by TV-image analysis in the same slides and plotted against time. Within practical limits, TV-image analysis using the P-curve gives the same results as the PLM method. Using the P-curve has the important advantage that its first part, beginning at the time of 3H-thymidine injection and ending at the first maximum, furnishes more information about the cell cycle than the corresponding part of the PLM curve. It can be used to compute tG2M tS and the ratio of the growth faction index to the cell-cycle time (IP/tC) whereas the first part of the PLM-curve reveals only the length of the S-phase (tS). The IP/tC ratio is a readily accessible measure of growth and increases when the cells divide more frequently. Cell death rates may be neglected since the ratio is determined within less than the duration of one cell cycle. Moreover, the data from the first part of the P curve indicate whether there is a large non-growth fraction. If the non-growth fraction is small, i.e. if IP approximately 1, the P curve need only be measured until the first maximum is reached so that fewer samples and animals are required. If the non-growth fraction is large or unknown, the cell-cycle parameters are calculated by reference to the position and size not only of the first minimum and the first maximum, but also of the second minimum of the P curve.     

Effect of nutritional insufficiency on cell proliferation in the matrix zones of the cerebellar anlage in mice

The work has been performed on 62 CBA mice. In the ventricular zone and in the external granular layer of the cerebellar anlage of embryos (13-17 days of the intrauterine development) mitotic index, labelled nuclei index, part of labelled mitoses have been counted. Parameters of the mitotic cycle of the matrix cells have been calculated by means of the graphic method. The proliferative pool value has been calculated. At malnutrition the cerebellar anlage structure retards in its maturation from the norm. For the matrix zones of the cerebellar anlage, higher indices of the proliferative activity are specific. At the same time, duration of the mitotic cycle of the matrix cells increases by 15-17%. It is possible, that retardation of histogenesis of the mouse cerebellar anlage, when developing under conditions of alimentary insufficiency depends on decreased rate of cell proliferation, as a result of prolonged mitotic cycle of the matrix cells.     

A rapid method to determine the mammalian cell cycle

A method was developed for determining the duration of the mammalian cell cycle and each of its major phases, mitosis, G1, DNA synthetic period, and G2. Mitotic time was determined by assessment of the mitotic index at intervals after cells collected in mitosis and stored at 4 °C were reincubated at 37 °C. The duration of the three remaining phases was derived from a graphic representation of the uptake of ³H-thymidine by a synchronous population of cells grown directly in scintillation vials. The scintillation counting method for determination of these parameters is advantageous over methods using autoradiography in that the investigator's bias in scoring cells is eliminated. Complex mathematical interpretations are unnecessary, and the data obtained from the scintillation counter are readily processed. Results from scintillation counting and autoradiographic methods are shown to be comparable.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.     

Analytical expressions for PLM(t), CL(t) and CLM(t) without use of Laplace transforms

Based on the age density functions for each phase of the cell life cycle (G₁, S, G₂ and M) in an exponentially growing steady state population derived by Trucco &; Brockwell (1968), the expressions for the percentage labeled mitoses curve [PLM(t)], the continuous labeling curve [CL(t)] and the continuous labeled mitotic curve [CLM(t)] are obtained explicitly without use of Laplace transforms. This approach is useful in describing the cell population when the steady state is disturbed due to, for example, irradiation. The mitotic index [MI(t)] for this case is considered.     

A new method for fish chromosome preparation

A new method for the preparation of fish chromosomes from abdominal cavity fluid has been developed. Cells were collected from fish abdominal cavity fluid after an in vivo PHA treatment, and cultured for a short time in medium with colchicine. After 30 min hypotonic treatment for marine fish and 35 min for freshwater fish, slides were prepared by the conventional air-drying method. The advantages ofthe method are: (1) it is technically simple; (2) it produces a reasonably high mitotic index; (3) chromosome spreading is good (4) there is very little cell breakage. Using this method, the chromosomes ofrainbow trout (2n=62); cod (2n=4546) and plaice (2n=46,47 and 48) were investigated.     

Determination of the mitotic index by microinjection of fluorescently labelled tubulin

The microneedle injection technique is one of the most established procedures for the introduction of proteins into living cells. To analyse injected proteins which are important in cell cycle progression it is often necessary to determine the mitotic index. Measuring the mitotic index after microinjection is complicated because only a limited number of cells of the whole cell population is microinjected. Therefore, we attempted to establish a new method to determine the mitotic index using microinjection of fluorescently labelled alpha/beta-tubulin into mammalian cells which allows to monitor the injected cells simultaneously with the determination of the mitotic index. We demonstrated that fluorescently labelled tubulin incorporates efficiently into the mitotic spindle apparatus. Fluorescence remains stable for several hours which is sufficient to observe the progression of cells through the M-phase of the cell cycle. The determination of the mitotic index with the method presented here gave similar results to those determined using other methods. With this method also different stages of mitosis can be visualized by analysing various steps of spindle formation. Thus, this rapid method allows the monitoring of the injected cells after microneedle injection and simultaneously the determination of the mitotic index.     

TURNOVER OF CELL-RENEWING POPULATIONS UNDERGOING CIRCADIAN RHYTHMS IN CELL PROLIFERATION

Evidence is presented in support of the concept of partial synchrony of cells as the cause for circadian rhythms in DNA synthesis and mitotic activity. The nonuniform age distribution of cells in cycle indicated that equations based on total asynchrony were not applicable for calculation of cytokinetic parameters in cellrenewing populations undergoing circadian rhythms. The integration of the circadian mitotic curve is introduced as a simple and accurate method for determination of proliferation rate and turnover time. An approximately linear increase in the labeling index following repeated injections of ³H-thymidine demonstrated that nearly 100% basal cells in hamster cheek pouch epithelia were in cycle during a turnover time. These experiments suggest that if there is a G₂ phase in cellrenewing tissues, this is short with respect to turnover time and that it may be a specific compartment where the control of cell proliferation operates.     

Circadian rhythms in the mitotic index of the basal epithelium and in the uptake rate of 3 H-thymidine by the tongue of the rat

Circadian rhythms are demonstrated in the tongue of adult rats for both the mitotic index of the basal epithelium and the uptake rate of injected 3H-thymidine by the tongue tip. The animals were entrained to a light-dark cycle for four weeks prior to the experiments with the light phase extending from 0600 to 1800 hours (CST). The daily fluctuation is approximately 300% for the mitotic index and 185% for the uptake rate of 3H-thymi-dine. The highest mitotic index occurs at 1100, and the highest uptake of 3H-thymidine occurs four hours earlier at 0700. The least activity for both parameters occurs during the first part of the dark span of the light-dark cycle. Estimates of several other rhythmic parameters are determined by a computerized method.     

Mitotic activity and its 24-hour rhythm in the rat pineal gland

Previous studies have yielded equivocal results concerning the 24-hour rhythmicity of mitotic activity in the rat pineal. The aim of the present study was to re-investigate this problem by carrying out three separate 24-hour experiments on alternate days. The results obtained confirm previous findings showing that in the pineal gland of adults mitotic activity is low. On average 22.3 mitotic figures of pinealocytes are seen per pineal gland, corresponding to a mitotic index of 0.2-0.6/1,000 pinealocytes. Mitotic activity is distinctly higher at daytime than at night. The timing of the peaks and troughs differs slightly from experiment to experiment. The majority of observations now indicate that in the rat pineal gland mitotic activity is higher at day time than at night.     

Calculation of the duration of mitosis for a population with an exponential growth of the cell number

A formula was received for the mean mitotic duration of a cell population being at the phase of exponential growth of the cell number with the cell loss: tM = nM.tD.(1-phi)/ln 2, where nM is the mitotic index, tD is the doubling time of the cell number, and phi is the Steel cell loss factor. In the case when after irradiation of such a population a 100% radiation G2-block arises, the method of calculation of the tM according to the curve of the relative mitotic index decrease was shown to be independent on the value of parameter phi and to coincide with the same method to be used in the case when the cell population is at the steady state before irradiation. As the result of analysis of literary experimental data the following values were received: tM = 20 min and tM = 37-42 min for two cell subpopulations of the Ehrlich ascite tumour, resp., tM = 39 min for epithelial cells of the mouse cornea, and tM = 29 min for enterocytes of the mouse jejunum. It has been also shown that the values of the dose of cell irradiation and of the mean duration tG2" of the subphase G2" localized at the end of phase G2 and preceded by the G2-block satisfy the next formula: Ig D = -a . Ig tG2" + b.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Rats were treated with guanethidine-sulphate every 48 hr from birth until 15 days and then maintained until young adulthood. Sympathectomy was verified by dissection and light microscopic preparation of the superior cervical and coeliac ganglia which showed at least a 78% reduction in the number of perikarya. The effect of the chemical sympathectomy was a decrease in the amplitude of the circadian mitotic rhythm from 44·7 to 27·1%, 67·0 to 25·3% and 54·9 to 24·7%, in the duodenum, jejunum, and ileum, respectively. The shape of the mitotic index curve was altered and the mean mitotic index was significantly decreased (P < 0·01) in all three segments of the small intestine. The mean mitotic index of control intestinal epithelium was 3·2 ± 0·1%, 3·6 ± 0·1% and 4·0 ± 0·1% in the duodenum, jejunum, and ileum, respectively, and 2·3 ± 0·1%, 2·4 ± 0·1%, and 2·5 ± 0·1% in guanethidine-treated rats. Stathmokinetic estimates of cycle time were obtained by use of the metaphase arrest agent, colchicine. The longest cell generation cycle time (T_c) and lowest mitotic index occurred between 12.00 and 16.00 hours and the shortest T_c and highest mitotic index occurred between 00.00 and 04.00 hours, in all three segments of the small intestine. Guanethidine-treatment lengthens T_c throughout the small intestinal epithelium and reduces the range of variation in T_c over a 24-hr period. It is suggested that norepinephrine depletion induced by guanethidine may be the cause of the inhibition in circadian periodicity and that norepinephrine and the sympathetic nervous system may be essential for the maintenance of circadian rhythms in mitotic activity.     

CELL DIVISION STUDIES OF THE SHOOT APEX OF DATURA STRAMONIUM DURING TRANSITION TO FLOWERING

Seedlings of Datura stramonium L., although not photoperiodically sensitive, are useful for floral transition studies when raised in a growth chamber at a constant temperature of 25 C with a photoperiod of 8 hr of light (1,600-2,000 ft-c) and 16 hr of darkness. A terminal flower is formed after the seventh or eighth leaf primordium is produced. A constant rate of leaf initiation up to the time of flowering enables specific apical stages to be obtained and studied. Changes in the mitotic index, substantiated with calculated rates of cell division (measured by the accumulation of metaphases following treatment with colchicine) were studied in shoot apical zones during transition to flowering. Fluctuations in the mitotic index of each zone in the vegetative and transition apex with respect to apical stage as well as time of day were not statistically significant. The mitotic index of the summit zone of the vegetative apex was significantly lower than in the other zones whose mitotic indices were not significantly different from one another. During floral transition the mitotic index of the summit zone as well as the central zone (just below the summit zone) significantly increased while no significant changes were detected in the flank zones. It was shown that the mitotic index could be considered representative of the rates of cell division in Datura.     

Measuring the mitotic index in chemically-treated human lymphocyte cultures by flow cytometry

In the human lymphocyte chromosome aberration assay, the mitotic index (MI) is the standard cytotoxic parameter for determining which test concentrations will be evaluated for chromosome aberrations. Assessment of the MI is performed microscopically by determining the frequency of mitotic cells in a population of 1000 cells. With the commercial availability of antibodies to the mitosis-specific marker, phosphorylated-histone H3 at serine 10, automating the assessment of the MI using flow cytometry is now possible [Cytometry 32 (1998) 71]. Our laboratory has utilized and validated this technology to measure the mitotic index of chemically-treated human lymphocyte cultures. Comparisons between the microscopic and flow MI frequencies from 24h treatments with mitomycin-C, aphidicolin, eugenol, etoposide, hydroxyrurea, potassium cyanide, staurosporine, ethyl alcohol, noscapine and colcemid((R)) are presented. Our results show that the mitosis specific H3-P marker is excellent for measuring the MI frequency in human lymphocyte cultures treated up to toxic concentrations. In addition, this study demonstrates that automation of analysis by flow cytometry is an excellent alternative to the microscopic method of analysis producing less variability than the microscopic scoring and a more complete dose response curve.     

THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH*

The initial effect of an injection of TdR-5-³H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G₂-cells at the time of injection. The main effect is a shortening of t_g2. No effect is observed after injection of non-radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with ³H-TdR, as well as the risks of using the PLM-method in a partially synchronized system.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.