scholarly journals Fluorescence polarization and pulse width analysis of chromosomes by a flow system.

1979 ◽  
Vol 27 (1) ◽  
pp. 445-453 ◽  
Author(s):  
L S Cram ◽  
D J Arndt-Jovin ◽  
B G Grimwade ◽  
T M Jovin
Keyword(s):  
Dna Content ◽  
Pulse Width ◽  
Flow System ◽  
Chinese Hamster ◽  
Hoechst 33342 ◽  
Cell Sorter ◽  
Emission Anisotropy ◽  
Simple Theoretical Model ◽  
Rigid Rods ◽  
Computer Controlled

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.

scholarly journals DNA analysis and sorting of viable mouse testis cells.

1981 ◽  
Vol 29 (6) ◽  
pp. 738-746 ◽  
Author(s):  
W M Grogan ◽  
W F Farnham ◽  
J M Sabau
Keyword(s):  
Dna Content ◽  
Adult Mouse ◽  
Dna Analysis ◽  
Cell Types ◽  
Mouse Testis ◽  
Spermatogenic Cells ◽  
Hoechst 33342 ◽  
Cell Sorter ◽  
Pachytene Spermatocytes

The dye Hoechst 33342 and a 2-parameter cell sorter have been used to measure DNA content in viable testis cells and to sort pachytene spermatocytes and round spermatids from adult mouse testis to virtually 100% homogeneity. Early diploid spermatogenic cells were enriched to 90%, a 10-fold purification. The capability for viable sorting of most testis cell types to homogeneity in numbers suitable for many biochemical applications is demonstrated.

scholarly journals Analysis and sorting of living cells according to deoxyribonucleic acid content.

1977 ◽  
Vol 25 (7) ◽  
pp. 585-589 ◽  
Author(s):  
D J Arndt-Jovin ◽  
T M Jovin
Keyword(s):  
Cell Cycle ◽  
Acid Content ◽  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
Acid Metabolism ◽  
Flow System ◽  
Living Cells ◽  
Hoechst 33258 ◽  
Cell Sorter ◽  
Computer Controlled

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.

Past, present and future perspectives on sexing sperm

2003 ◽  
Vol 83 (3) ◽  
pp. 375-384 ◽  
Author(s):  
D. L. Garner ◽  
G. E. Seidel Jr.
Keyword(s):  
Flow Cytometry ◽  
Key Words ◽  
Sex Chromosomes ◽  
Dna Content ◽  
Sex Chromosome ◽  
Future Perspectives ◽  
Hoechst 33342 ◽  
Cell Sorter ◽  
Mammalian Sperm ◽  
Bovine Sperm

Development of flow cytometry for sorting mammalian sperm according to their sex chromosomes began in the late 1970s and early 1980s. This technology, which has recently been commercialized for bovine sperm, is based on the differences in DNA content between X- and Y-chromosome-bearing sperm. Under ideal conditions, 5000 live bovine sperm of each sex can be sorted per second at 90% accuracy. Pregnancy rates of 50% have been achieved routinely in well-managed heifers with sex-sorted, cryopreserved bovine sperm compared to 60–80% with unsexed control sperm. About 90% of offspring have been of the selected sex. Sorting sperm according to sex chromosome content is similarly successful in many other mammals including exotic species, but sorting efficiencies are somewhat less for sperm from some species. Key words: Mammals, sex chromosomes, flow cytometer, cell sorter, DNA content, X and Y sperm, Hoechst 33342

scholarly journals Cytotoxicity, Mutagenicity and DNA damage by Hoechst 33342.

1982 ◽  
Vol 30 (2) ◽  
pp. 111-116 ◽  
Author(s):  
R E Durand ◽  
P L Olive
Keyword(s):  
Dna Content ◽  
Chinese Hamster ◽  
Normal Growth ◽  
Living Cells ◽  
Good Resolution ◽  
V79 Cells ◽  
Hoechst 33342 ◽  
Efficient Inhibitor ◽  
Synthetic Rate ◽  
Coefficients Of Variation

Hoechst 33342 can be used for flow microfluorimetry (FMF) analysis of the DNA content of living Chinese hamster cells, giving good resolution (coefficients of variation (CVs) 6%) with relatively nontoxic staining regimens. The dye is, however, a very efficient inhibitor of DNA synthesis, with a marked depression of the DNA synthetic rate for V79 cells observed at concentrations tenfold less than those required for optimal FMF resolution. Nontoxic and minimally toxic Hoechst concentrations also resulted in demonstrable mutation, as assayed by 6-thioguanine resistance. Hoechst disappearance from cells returned to normal growth medium after staining suggests two components of binding, since about half of the total stain is rapidly removed, whereas the rest is apparently diluted only by cell division. Cells containing Hoechst 33342 die more rapidly than control cells when held at 4 degrees C and are also more susceptible to inactivation by the ultraviolet laser beam if operated at approximately greater than 100 mW power. Thus, Hoechst 33342 can provide information about the DNA content of living cells, but at the expense of minimal toxicity, moderate mutation, and significant cell cycle perturbations.

Study of the film formation process in liquid-liquid extraction flow injection analysis

1991 ◽  
Vol 56 (6) ◽  
pp. 1228-1237 ◽  
Author(s):  
Vlastimil Kubáň
Keyword(s):  
Flow System ◽  
Detection System ◽  
Film Formation ◽  
Liquid Extraction ◽  
Photometric Detection ◽  
Liquid Liquid Extraction ◽  
Teflon Tube ◽  
Continuous Stream ◽  
Computer Controlled ◽  
Segmented Volume

The behaviour of a thin film of an organic solvent on the walls of the extraction coil in a continuous liquid-liquid extraction flow system was studied using a computer-controlled fast-recording on-tube photometric detection system (approx. 3 ms time resolution). A single-loop injector was employed to introduce precise, reproducible volumes (Sr < 2%) of one phase into the continuous stream of the other as a segmented volume standard. The film thickness Df, ranging from 1 to 20 μm for a 0.7 mm teflon tube, was calculated from the segment lengthening at a different chloroform flow rates and was found to obey a polynominal dependence on the linear flow rate, df = f(uα), where α < 1.

scholarly journals Photoselection of Luminescent Molecules in Anisotropic Media in the Case of Two-Photon Excitation. Part II. Experimental Studies of Hoechst 33342 in Stretched Polyvinyl alcohol) Films

1996 ◽  
Vol 51 (9) ◽  
pp. 1037-1041 ◽  
Author(s):  
A. Kawski ◽  
Z. Gryczyński ◽  
I. Gryczyński ◽  
J. R. Lakowicz ◽  
G. Piszczek
Keyword(s):  
Polyvinyl Alcohol ◽  
Vinyl Alcohol ◽  
Experimental Studies ◽  
Poly Vinyl Alcohol ◽  
Hoechst 33342 ◽  
Emission Anisotropy ◽  
Two Photon ◽  
Photon Excitation ◽  
Polyvinyl Alcohol Films ◽  
Two Photon Excitation

Abstract It was found by investigating dichroism and emission anisotropy in the case of one-and two-photon excitation of Hoechst 33342 [bis-benzimide,2,5'-bi-1H-benzimidazole, 2'-(4-ethoxyphenyl)-5-5(4-methyl-1-piperazinyl)] in stretched poly(vinyl alcohol) (PVA) films, that the absorption and fluorescence transition moments lie along the long molecular axis of the molecule studied. The slight deviation of the transition moment direction in fluorescence (about 8°) from that in absorption can be due to the incomplete linearity of the Hoechst molecule.

scholarly journals Polyploid Formation via Chromosome Duplication Induced by CTP:Phosphocholine Cytidylyltransferase Deficiency and Bcl-2 Overexpression: Identification of Two Novel Endogenous Factors

2005 ◽  
Vol 53 (6) ◽  
pp. 725-733 ◽  
Author(s):  
You-Jun Shen ◽  
Cynthia J. DeLong ◽  
Francois Tercé ◽  
Timothy Kute ◽  
Mark C. Willingham ◽  
...  
Keyword(s):  
Dna Content ◽  
Chinese Hamster ◽  
B Cell Lymphoma ◽  
Negative Regulator ◽  
Ovary Cell ◽  
Endogenous Factors ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Polyploid Formation ◽  
Cellular Dna ◽  
Diploid Cells

Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CTα prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CTα as a negative regulator of polyploid formation.

Studies on sorted x and y enriched populations of bull spermatozoa

1993 ◽  
Vol 1993 ◽  
pp. 156-156
Author(s):  
N.G.A. Miller ◽  
E.A. Howes ◽  
D.G. Cran ◽  
L.A. Johnson
Keyword(s):  
Surface Charge ◽  
Y Chromosome ◽  
Dna Content ◽  
Egg Yolk ◽  
Flow Cytometer ◽  
Hoechst 33342 ◽  
Sample Stream ◽  
Inlet Tube ◽  
Specific Orientation ◽  
Cattle And Sheep

There have been many attempts to separate X- and Y-chromosome bearing spermatozoa on the basis of surface charge or density (Schröder, 1941; Bhattacharya et at., 1966; Engelman et al., 1988; Blottner et al., 1992). These have not met with universally accepted success. More recently, sorting has been achieved using the flow cytometer (FACS) to discriminate between the very small differences (3-4%) in DNA content of X- and Y-bearing spermatozoa (Johnson et al., 1989; Cran et al., 1993). However, the spermatozoa must be ‘flat headed’ e.g. rabbit, chinchilla, horse, pig, cattle and sheep.The flow cytometer (fitted with a U.V. laser) was adapted to accept a bevelled inlet tube. This gave us a ‘ribbon-like’ profile of the sample stream which forced the spermatozoa into a specific orientation.Semen was collected from healthy bulls of proven fertility and within 1/2 hr. was diluted to 1 x 107 per ml. and stained with Hoechst 33342 (5-10/μm) for 1 hr. at 35°C. Only those spermatozoa emerging from the 70μ nozzle that were edge-on to the 90° detector were selected as X or Y by the 0° detector. Gated intact spermatozoa were positioned into individual drops and then charged positive or negative for later deflection and collection into tubes containing egg yolk medium.

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