scholarly journals Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes.

1977 ◽  
Vol 25 (8) ◽  
pp. 954-964 ◽  
Author(s):  
R H Jensen ◽  
R G Langlois ◽  
B H Mayall
Keyword(s):  
Flow Cytometry ◽  
Energy Transfer ◽  
Ethidium Bromide ◽  
Deoxyribonucleic Acid ◽  
Fluorescent Dyes ◽  
Chinese Hamster ◽  
Fluorescence Properties ◽  
Chromomycin A3 ◽  
Metaphase Chromosomes ◽  
Mitotic Cells

Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.

scholarly journals Interactions between pairs of DNA-specific fluorescent stains bound to mammalian cells.

1979 ◽  
Vol 27 (1) ◽  
pp. 72-79 ◽  
Author(s):  
R G Langlois ◽  
R H Jensen
Keyword(s):  
Energy Transfer ◽  
Ethidium Bromide ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Fluorescence Emission ◽  
Hoechst 33258 ◽  
Mitotic Chromosomes ◽  
Chromomycin A3 ◽  
Efficient Energy Transfer ◽  
Fluorescence Emission Spectra

The interactions between DNA-specific fluorescence stains complexed with mitotic Chinese hamster cells were studied by spectrofluorometric and flow fluorometric techniques. The degree of binding interactions and of energy transfer between stains was determined from the intensities and shapes of fluorescence emission spectra of cells complexed with pairs of stains. The stain pairs Hoechst 33258-chromomycin A3, Hoechst 33258-ethidium bromide, and chromomycin A3-ethidium bromide exhibited efficient energy transfer from the short wavelength absorber (donor) to the long wavelength absorber (acceptor), and little competitive or cooperative binding of stains. The stain pair quinacrine-ethidium bromide exhibited both energy transfer and competitive binding. None of the stain pairs showed evidence of strong electronic interactions between stains. The magnitude of energy transfer interactions was used to estimate the quantity and distribution of the stains molecules complexed to mitotic cells. The results indicate a fairly even distribution of each of these stains along the DNA of intracellular mitotic chromosomes.

scholarly journals Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.

1977 ◽  
Vol 25 (7) ◽  
pp. 927-934 ◽  
Author(s):  
S A Latt ◽  
Y S George ◽  
J W Gray
Keyword(s):  
Flow Cytometry ◽  
Cell Growth ◽  
Deoxyribonucleic Acid ◽  
Flow Cytometric Analysis ◽  
Chinese Hamster ◽  
Acid Synthesis ◽  
Whole Cells ◽  
Flow Cytometric ◽  
Deoxyribonucleic Acid Synthesis ◽  
Average Fluorescence

This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.

scholarly journals Slit-scan flow cytometry of mammalian chromosomes.

1979 ◽  
Vol 27 (1) ◽  
pp. 441-444 ◽  
Author(s):  
J W Gray ◽  
D Peters ◽  
J T Merrill ◽  
R Martin ◽  
M A Van Dilla
Keyword(s):  
Flow Cytometry ◽  
Laser Beam ◽  
Ethidium Bromide ◽  
Chinese Hamster ◽  
Flow Cytometer ◽  
Total Fluorescence

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.

scholarly journals Pairs of fluorescent dyes as probes of DNA and chromosomes.

1979 ◽  
Vol 27 (1) ◽  
pp. 65-71 ◽  
Author(s):  
S A Latt ◽  
E Sahar ◽  
M E Eisenhard
Keyword(s):  
Energy Transfer ◽  
Base Composition ◽  
Fluorescent Dyes ◽  
Structural Features ◽  
The Other ◽  
Base Pairs ◽  
Metaphase Chromosomes ◽  
Energy Donor ◽  
Chromosome Regions ◽  
Energy Acceptor

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.

scholarly journals Fluorescent DNA probes for flow cytometry. Considerations and prospects.

1979 ◽  
Vol 27 (12) ◽  
pp. 1652-1654 ◽  
Author(s):  
H A Crissman ◽  
D J Orlicky ◽  
R J Kissane
Keyword(s):  
Flow Cytometry ◽  
Energy Transfer ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
Control Measures ◽  
Fluorescence Measurements ◽  
Base Specificity ◽  
Associated Proteins ◽  
Fluorescent Compounds

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.

scholarly journals Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

1977 ◽  
Vol 25 (7) ◽  
pp. 573-579 ◽  
Author(s):  
R H Jensen
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Deoxyribonucleic Acid ◽  
Emission Spectra ◽  
Hoechst 33258 ◽  
Chromomycin A3 ◽  
Fluorescence Excitation ◽  
Specific Staining ◽  
Cervical Cells ◽  
Two Parameter

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.

Efficient artificial light-harvesting systems based on aggregation-induced emission constructed in supramolecular gels

Soft Matter ◽  
2021 ◽  
Author(s):  
Xinxian Ma ◽  
bo qiao ◽  
Jinlong Yue ◽  
JingJing Yu ◽  
yutao geng ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Rhodamine B ◽  
Fluorescent Dyes ◽  
Light Harvesting ◽  
Artificial Light ◽  
Efficient Energy Transfer ◽  
Aggregation Induced Emission ◽  
Efficient Energy ◽  
Harvesting Systems ◽  
Acyl Hydrazone

Based on a new designed acyl hydrazone gelator (G2), we developed an efficient energy transfer supramolecular organogel in glycol with two different hydrophobic fluorescent dyes rhodamine B (RhB) and acridine...

scholarly journals KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

1970 ◽  
Vol 47 (3) ◽  
pp. 689-702 ◽  
Author(s):  
Hartmut C. Renger ◽  
David R. Wolstenholme
Keyword(s):  
Electron Microscope ◽  
Ethidium Bromide ◽  
Thermal Denaturation ◽  
Deoxyribonucleic Acid ◽  
Cesium Chloride ◽  
Kinetoplast Dna ◽  
Microscope Examination ◽  
Main Band ◽  
Trypanosoma Lewisi ◽  
High Degree

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.

scholarly journals Membrane traffic between secretory compartments is differentially affected during mitosis.

1990 ◽  
Vol 1 (5) ◽  
pp. 415-424 ◽  
Author(s):  
T Kreiner ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Traffic ◽  
Human Growth ◽  
Secretory Proteins ◽  
Viral Membrane ◽  
Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus

1981 ◽  
Vol 1 (3) ◽  
pp. 237-244
Author(s):  
D B Yarosh ◽  
R B Setlow
Keyword(s):  
Polyethylene Glycol ◽  
Deoxyribonucleic Acid ◽  
Excision Repair ◽  
Micrococcus Luteus ◽  
Chinese Hamster ◽  
Endonuclease Activity ◽  
Chinese Hamster Cells ◽  
Ultraviolet Resistance

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

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