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191.
Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma. 相似文献
192.
A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro 总被引:4,自引:0,他引:4
Timothy A. McCaffrey Lily A. Agarwal Babette B. Weksler 《In vitro cellular & developmental biology. Plant》1988,24(3):247-252
Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate,
time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts.
The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence.
Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in
a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods.
The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced
growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation
assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA.
This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is
necessary.
This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging
Research to T. M. and HL35724 to B. W.
EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers
of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells. 相似文献
193.
Allison A. Welder Tina Machu Steven W. Leslie Richard E. Wilcox June Bradlaw Daniel Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):771-777
Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models
for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding,
beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained
from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude
membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B
max
(42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K
D
of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after
3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7
M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess
saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable
than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology
Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration. 相似文献
194.
Kiyoshi Akeo Yasuhiko Tanaka Tatsuji Fujiwara 《In vitro cellular & developmental biology. Plant》1988,24(7):705-710
Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin.
Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and
coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm
after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of
membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside
the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation,
appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules
and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small
ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around
or in the Golgi apparatus.
Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986. 相似文献
195.
A. Birkenfeld Y. Ezra N. Ron D. Navot S. Granovsky J. G. Schenker I. S. Levij I. Vlodavsky 《In vitro cellular & developmental biology. Plant》1988,24(12):1188-1192
Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a
growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial
basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix
(ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on
ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like
cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission
electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which
ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping,
closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type
specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane
antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial
component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture
dishes.
This work was supported by PHS grant no. CA 30289 to J.V. 相似文献
196.
197.
Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2 总被引:1,自引:0,他引:1
L. E. J. Lee A. Martinez N. C. Bols 《In vitro cellular & developmental biology. Plant》1988,24(8):795-802
Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined
and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown
to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures.
Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After
3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of
fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which
either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum
deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined.
This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation
should allow the G1-S transition to be studied in a representative of teleosts.
This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B. 相似文献
198.
Francesca Solanellas Albert Bordons 《Journal of industrial microbiology & biotechnology》1988,3(4):205-209
Summary A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested. 相似文献
199.
Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed. 相似文献
200.
F Puvion-Dutilleul E Pichard 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,76(2):139-150
Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication. 相似文献