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Comparison of the cell kill measured by the Hoechst-propidium iodide flow cytometric assay and the colony formation assay. hoechst -碘化丙啶流式细胞术与菌落形成法测定细胞杀伤的比较。
Pub Date : 1983-07-01
C A Wallen, R Higashikubo, J L Roti Roti

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.

使用Hoechst 33342和碘化丙啶(HO-PI)双染色的流式细胞术活细胞测定法评估了其确定处理HeLa细胞克隆原性的能力。集落形成试验被用作参考,以确定HO-PI试验的能力,以测量在给定群体中存在的克隆生成细胞的比例。流式细胞术和台盼蓝染色排除试验的活力估计都能准确预测未经处理的HeLa细胞群体的集落形成效率(CFE)。然而,在加热、冻融或电离辐射处理后,HO-PI实验大大高估了HeLa细胞的克隆原性。当细胞被加热(45℃,30分钟),然后在处理后0,1,2,3,6,12,16或23小时进行检测时,也观察到FCM测定的活力与克隆性存活之间缺乏相关性。这些数据表明,HO-PI所估计的生存能力并不能预测急性治疗后的生存。在67只小鼠乳腺肿瘤细胞中,当克隆原性随着营养剥夺条件(慢性治疗)时间的延长而降低时,HO-PI测定再次预测了比测量值更高的CFEs。因此,在这些实验中,HO-PI法不能预测细胞死亡,也不能比台盼蓝染料排除试验更好地衡量细胞活力。
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引用次数: 0
Stathmokinetic measurement of tumour cell proliferation in relation to vascular proximity. 肿瘤细胞增殖与血管接近的静态动力学测量。
Pub Date : 1983-07-01
B Jones, R S Camplejohn

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.

在这项研究中,使用长春新碱(VCR)中期阻滞方法,在移植的C3H小鼠乳腺肿瘤中,研究细胞增殖与血管距离的关系。中期阻滞方法与涉及[3H]TdR标记的方法相比具有许多优点。细胞出生率(kB)显示与肿瘤有绳区毛细血管的距离成反比。穿透VCR,甚至进入肿瘤的会阴坏死区域,似乎足以实现有效的中期抑制。
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引用次数: 0
Autoradiographic study of colchicine inhibition of DNA synthesis and cell migration in hairless mouse epidermis in vivo. 秋水仙碱抑制小鼠无毛表皮DNA合成及细胞迁移的放射自显影研究。
Pub Date : 1983-07-01
B Epstein, J H Epstein, K Fukuyama

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.

在体内研究了秋水仙碱对无毛小鼠表皮细胞有丝分裂计数、DNA合成和细胞迁移时间的影响。[H3]TdR检测DNA合成并测定传递时间。我们发现秋水仙碱(剂量为50微克/25克体重)引起有丝分裂停止,但对注射后4小时内处于S期的细胞数量没有影响。在注射后16-30小时,秋水仙碱可减少正常表皮S期细胞的数量,但在注射后42小时,这种作用不复存在。在注射秋水仙碱48小时后,uvr刺激的增生表皮中出现了类似的s期细胞计数下降。此外,秋水仙碱可干扰uvr刺激下增生性表皮[H3] tdr标记细胞从基底层向浅层的迁移。运动延迟在前24小时内未见,但在注射后48小时出现。
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引用次数: 0
Cloning and frequency analysis of haemopoietic stem cells producing CFC over weeks in long-term marrow cultures. 在长期骨髓培养中产生CFC的造血干细胞的克隆和频率分析。
Pub Date : 1983-07-01
H G Mergenthaler, F G Staber, P Dörmer

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.

利用无干细胞的贴壁骨髓细胞层,通过限制稀释的方法进行体外克隆和小鼠造血干细胞频率的测定。干细胞在个体微培养中的存在通过体外集落形成细胞(CFC)的持续存在间接证明。这些细胞的数量随着培养时间的增加而增加,这似乎表明CFC是在体外从头产生的。虽然在一些实验中确定了与体内脾脏集落试验相当的干细胞频率,但我们的大多数频率估计表明干细胞频率范围为5/10(6)至90/10(6)。大约4周后,干细胞活性在微培养中迅速下降。这可能是由脑脊液和/或类似因素控制的分化压力增加或次优培养条件引起的。
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引用次数: 0
Autoradiographic studies of glial proliferation in different areas of the brain of the 14-day-old rat. 14日龄大鼠大脑不同区域胶质细胞增殖的放射自显影研究。
Pub Date : 1983-07-01
H Korr, W D Schilling, B Schultze, W Maurer

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.

采用不同的细胞动力学方法研究了注射[3H]TdR和(或)[14C]TdR后,14日龄大鼠大脑皮层、胼胝体、壳核尾状核和前联合四个不同区域的细胞周期参数以及胶质细胞的增殖模式。S期持续时间(tS)约为10小时,周期时间(tC)约为20小时,tG2小于2小时,t(G2 + M)约为4小时。这些值对研究的所有四个脑区的神经胶质细胞都有效。然而,神经胶质细胞的标记指数(LI)在不同脑区相差3倍,在1.8到5.4%之间。相应的,四个区域的胶质细胞群的生长分数在0.04 ~ 0.12之间变化。神经胶质细胞(星形胶质细胞和少突胶质细胞)按照一个稳定的状态系统增殖。此外,胶质细胞的增殖与持续的细胞损失有关。每次有丝分裂后,约有3%的子细胞收缩并死亡。此外,胶质细胞的永久交换发生在增殖池和非增殖池之间。
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引用次数: 0
A comparison of the cell kinetics of pre-implantation mouse embryos from two different mouse strains. 两种不同小鼠品系植入前小鼠胚胎的细胞动力学比较。
Pub Date : 1983-05-01
M Molls, N Zamboglou, C Streffer

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.

用细胞荧光法研究了小鼠胚胎着床前的第一、第二和第三个胚胎细胞周期。与以往大多数研究不同的是,卵子是自发排卵的,卵子供体的交配期较短(06.00-09.00小时)。胚胎细胞作为一个队列经历了第一、第二和第三细胞周期。因此,可以直接从DNA直方图估计细胞周期阶段的持续时间。细胞周期的长度在两种不同小鼠品系的胚胎之间存在差异。在G2 + M期差异最明显(菌株I和菌株II的第一个细胞周期分别为8小时和5小时;第二个细胞周期:分别为11.5小时和14小时)。然而,根据以往的研究,还观察到早期植入前细胞动力学的共同特征:从第一个细胞周期到第二个细胞周期的S期长度增加,第二和第三个细胞周期的G1期非常短。在第三个细胞周期后,两种菌株的胚胎细胞增殖均呈指数增长。菌株I胚的增殖率高于菌株II胚(生长曲线的上升幅度更大)。在着床前发育(胚泡孵化)结束时,两种菌株的生长曲线均下降。有关细胞周期阶段的持续时间和增殖率的差异被认为是菌株特异性的。这表明,已经描述的着床前细胞动力学的差异通常反映了菌株特异性,而不是不同的研究方法和/或不同等级的早期着床前胚胎的同步。
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引用次数: 0
Survival of transfused cryopreserved granulocytic progenitor cells (CFU-C) in recipient circulation. 输注低温保存的粒细胞祖细胞(CFU-C)在受体循环中的存活。
Pub Date : 1983-05-01
A Raghavachar, K H Steinbach, O Prümmer, G Grilli, T M Fliedner

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.

我们研究了在自体和异体供体-受体关系下,给予骨髓、胎儿肝脏和外周血中含有已知数量的CFU-C的冷冻保存细胞悬液的正常和全身辐照犬的外周血中CFU-C消失的模式。在输注结束时,在受体循环中只能检测到一小部分输注的供者CFU-C。循环CFU-C呈指数下降,提示输注CFU-C随机丢失。各实验组CFU-C消失模式具有可重复性。正常受者循环血液中自体血源性CFU-C的平均半衰期为8.2 min,计算血液中CFU-C的平均周转量为9.3 × 10(5) CFU-C/kg / d。
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引用次数: 0
A simple method for analysis of DNA histograms in human myeloid cells perturbed by stimulators of DNA synthesis. DNA合成刺激物干扰的人髓细胞DNA直方图的简单分析方法。
Pub Date : 1983-05-01
M B Henderson, S M Myers, P R Galbraith

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.

在过去,相分馏已被用作确定受DNA合成刺激物和抑制剂干扰的细胞的刺激水平。在我们手中,这种方法被证明对人类骨髓细胞不敏感,也不可靠。本文描述了一种新的分析方法,该方法涉及利用S期中期拟合线的斜率大小作为培养的人骨髓细胞刺激水平的指标。该方法快速、可靠、简单,可在廉价的微机上实现。
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引用次数: 0
Automated radioautographic grain counting. Correction for grain overlap. 自动放射自动计数。修正颗粒重叠。
Pub Date : 1983-05-01
W H Schuette, S S Chen, S J Occhipinti, H S Mujagic, S E Shackney

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.

描述了一种通过测量总细胞核面积和总粒面积计算放射自显影细胞粒数的算法。该算法基于概率论中占位问题的求解,对颗粒重叠进行统计校正。该方法允许在每个细胞的谷物计数范围内使用自动谷物计数,并将放射自显影谷物计数的有用动态范围扩展到超过200粒/细胞。
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引用次数: 0
A quantitative evaluation of pulmonary macrophage kinetics. 肺巨噬细胞动力学的定量评价。
Pub Date : 1983-05-01
A Blussé van Oud Alblas, H Mattie, R van Furth

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.

一种新的数学方法来计算动力学的巨噬细胞在一个组织室提出。这种方法考虑了单核细胞流入腔室、单核吞噬细胞局部分裂和巨噬细胞从腔室流出的情况,并应用于正常稳定状态下小鼠肺巨噬细胞的数据。结果表明,至少70%的肺巨噬细胞群由单核细胞内流提供,最多30%由来自骨髓的未成熟单核吞噬细胞的局部分裂提供。计算出肺巨噬细胞的周转时间约为6天,周转量为14.6 × 10(3)个巨噬细胞/小时。
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引用次数: 0
期刊
Cell and tissue kinetics
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