scholarly journals Measurement of cellular DNA mass by flow microfluorometry with use of a biological internal standard.

1978 ◽  
Vol 26 (2) ◽  
pp. 145-148 ◽  
Author(s):  
E Tannenbaum ◽  
M Cassidy ◽  
O Alabaster ◽  
C Herman
Keyword(s):  
Dna Content ◽  
Internal Standard ◽  
Cell Populations ◽  
Experimental Cell ◽  
Flow Microfluorometry ◽  
Chicken Erythrocytes ◽  
Cellular Dna ◽  
Biological Standard

Use of a biological standard (chicken erythrocytes) mixed with experimental cell populations allows control of all variables in flow microfluorometric determination of DNA content. These variables include both staining and instrument procedures. In addition, the use of a biological standard allows determination of cellular DNA mass in unperturbed cell populations. DNA mass measured by FMF technique correlates closely with values reported in the literature that used biochemical techniques.

Genetic instability of multiple buds of Pinuscoulteri regenerated from tissue culture

1982 ◽  
Vol 12 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Kamlesh R. Patel ◽  
Graeme P. Berlyn
Keyword(s):  
Nuclear Dna ◽  
Internal Standard ◽  
Progressive Increase ◽  
Culture Conditions ◽  
Dna Contents ◽  
Large Populations ◽  
Multiple Buds ◽  
Chicken Erythrocytes ◽  
Cellular Dna ◽  
Over Time

Embryo expiants ofPinuscoulteri D. Don. were cultured on agar media (modified from Campbell and Durzan 1975) only supplemented with 2.25 mg•L−1 of benzyl amino purine (BAP). Nuclear DNA contents were measured in cells of the embryo explants, callus, and buds regenerated from these explants, and sand-germinated seedlings. Measurements were made at weekly intervals during 6 weeks of culture. Absolute amounts of cellular DNA were determined with a microspectrophotometer using chicken erythrocytes as an internal standard. Initially, the nuclei had a DNA level between 2C and 4C with a mean of 3C. The sand-grown seedlings remained within this range, with an increased frequency of 4C nuclei in the 6-week seedlings. However, cells from callus and regenerated buds showed a progressive increase in DNA level over time. After 42 days in culture, the buds contained large populations of cells at the 8C level and a considerable number of cells with DNA levels above 8C. In addition, the nuclei showed a considerable increase in chromosomal aberrations including bridges, micronuclei, lagging chromosomes, and fragmentation. Nuclear volume also increased over time in the regenerated buds. It is therefore concluded that culture conditions, perhaps cytokinin content, increased the levels of nuclear DNA. These increased levels could be the result of polyploidy, polyteny, hyperaneuploidy, or a combination of these types of nuclear organization.

scholarly journals NUCLEAR DNA CONTENT OF DENDROBIUM ORCHID SPECIES AS DETERMINED BY LASER FLOW CYTOMETRY

HortScience ◽  
1996 ◽  
Vol 31 (3) ◽  
pp. 322c-322
Author(s):  
W.E. Jones ◽  
A.R. Kuehnle ◽  
K. Arumuganathan
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Nuclear Dna ◽  
Evolutionary Relationship ◽  
Internal Standard ◽  
Nuclear Dna Content ◽  
Orchid Species ◽  
Group Assignment ◽  
Taxonomic Groups

Flow cytometry (FC) has proven to be an efficient and reliable method to estimate nuclear DNA content (genome size) in quantifiable units useful for genetic and molecular biology studies. This method also makes possible determination of the variation in nuclear DNA content between related taxa, which gives insights into the process of speciation. In this study, DNA content was determined in nuclei isolated from leaves of 21 Dendrobium species representing each of the major taxonomic groups used in the Univ. of Hawaii breeding program. Nuclei were mechanically isolated, stained with the nucleic acid-specific fluorochrom propidium iodide, and DNA content determined using a Coulter Epics 753 laser flow cytometer. Chicken erythrocyte nuclei (2C = 2.33 pg DNA) were used as an internal standard for direct comparative measurement. The mean diploid genome (2C) values for Dendrobium species ranged from 3.36 to 5.06 pg. Genome sizes were evaluated for possible use as discrete characters for taxonomic group assignment and compared to previous data on breeding compatibility and evolutionary relationship between species.

scholarly journals Flow microfluorometric quantitation of the blastogenic response of lymphocytes.

1976 ◽  
Vol 24 (1) ◽  
pp. 383-387 ◽  
Author(s):  
L S Cram ◽  
E R Gomez ◽  
C O Thoen ◽  
J C Forslund ◽  
J H Jett
Keyword(s):  
High Resolution ◽  
Dna Content ◽  
Deoxyribonucleic Acid ◽  
Low Salt ◽  
Blastogenic Response ◽  
Flow Microfluorometry ◽  
Specific Stimulation ◽  
Cellular Dna ◽  
Bovine Lymphocytes ◽  
Stimulation Of

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.

Flow cytometric determination of DNA content in isolated nuclei of cereals

Genome ◽  
1988 ◽  
Vol 30 (4) ◽  
pp. 565-569 ◽  
Author(s):  
E. Hülgenhof ◽  
R. A. Weidhase ◽  
R. Schlegel ◽  
A. Tewes
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Internal Standard ◽  
Aegilops Speltoides ◽  
Isolated Nuclei ◽  
Triticum Timopheevi ◽  
Flow Cytometric ◽  
Nuclear Isolation ◽  
Cereal Plants

Isolated nuclei from cereal plants were used for quantitative determination of DNA per nucleus by flow cytometry. The technique is based on enhanced fluorescence of ethidium bromide and olivomycin when they bind to DNA. Nuclei were isolated from protoplasts derived from leaves of seedlings. The diploid Hordeum vulgare cv. Trumpf was used as an internal standard. Analysis of nuclei from several cultivars of hexaploid wheat (Triticum aestivum), Triticum durum var. hordeiforme, T. durum var. alexandrinum, Triticum araraticum, Triticum timopheevi, Triticum monococcum, Aegilops speltoides, Secale cereale, and one hexaploid and one octoploid triticale revealed significant intra- and inter-specific differences in DNA values per 2C nucleus. The advantages of the procedure are discussed along with its utilization for quantitative DNA measurement in Gramineae.Key words: nuclear isolation, flow cytometry, DNA content, cereals.

scholarly journals Increased accuracy of absorption cytophotometric DNA values by control of stain intensity.

1981 ◽  
Vol 29 (10) ◽  
pp. 1219-1228 ◽  
Author(s):  
D C Allison ◽  
P F Ridolpho ◽  
E M Rasch ◽  
R W Rasch ◽  
T S Johnson
Keyword(s):  
Dna Content ◽  
Bone Marrow Cells ◽  
Internal Standard ◽  
Staining Reaction ◽  
Spleen Cells ◽  
Coefficients Of Variation ◽  
Rapid Calculation ◽  
Dna Values ◽  
Cellular Dna ◽  
Selection Of

A method of improving absorption cytophotometric cellular DNA values by making measurements on Feulgenstained cells at optimal stain absorbances has been developed. Stain intensity can be controlled either by alteration of the Feulgen staining reaction or by selection of "off-peak" wavelengths of light for cytometry. The use of chicken red blood cells as an internal standard, and of a computerized cytometer for the measurements, allows selection of the appropriate off-peak light wavelengths, correction for staining variability at different sites on the same slide, and rapid calculation of cellular DNA values. Cytometry can also be performed at controlled absorbance levels on autoradiographs of 3H-thymidine-labeled cells to allow direct study of the DNA content of nonlabeled G1/G0 and G2/M cells. Use of this technique on mixtures of mouse thymocytes, spleen cells, bone marrow cells, and liver cells gave essentially identical values for G1/G0 cellular DNA content, with coefficients of variation of less than 3%.

scholarly journals Nuclear DNA Content and Ploidy Levels in the Genus Ipomoea

1994 ◽  
Vol 119 (1) ◽  
pp. 110-115 ◽  
Author(s):  
Peggy Ozias-Akins ◽  
Robert L. Jarret
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Large Scale ◽  
Nuclear Dna ◽  
Internal Standard ◽  
Nuclear Dna Content ◽  
Ploidy Levels ◽  
Genetic Structure Of Populations ◽  
Chicken Erythrocytes ◽  
Nuclei Ploidy Level

The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei. Ploidy level and DNA content were significantly correlated within the genus, but more highly so within species that contained multiple cytotypes. DNA content of cultivated Z. batatas (L.) Lam. (4.8 to 5.3 pg/2C nucleus) and feral tetraploid I. batatas (3.0 to 3.5 pg/2C nucleus) was estimated from the known DNA content of chicken erythrocytes (2.33 pg), which were used as an internal standard. Tetraploid forms of Z. cordato-triloba Dennstedt also were identified. Ploidy analysis using flow cytometry is rapid and suitable for large-scale experiments such as studying the genetic structure of populations of Z. batatas and related species. Chemical name used: 4′,6-diamidino-2-phenylindole (DAPI).

scholarly journals Comparison of image analysis and flow cytometric determination of cellular DNA content.

1991 ◽  
Vol 44 (2) ◽  
pp. 147-151 ◽  
Author(s):  
C Cope ◽  
D Rowe ◽  
L Delbridge ◽  
J Philips ◽  
M Friedlander
Keyword(s):  
Image Analysis ◽  
Dna Content ◽  
Flow Cytometric ◽  
Cellular Dna

Determination of cellular DNA content of Iberian salamanders by flow cytometry

2000 ◽  
Vol 21 (4) ◽  
pp. 411-418 ◽  
Author(s):  
Miguel Lizana ◽  
Roberto Martín-Sánchez ◽  
Rafael Márquez ◽  
Juana Ciudad ◽  
Antonio López ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Reproductive Biology ◽  
Dna Content ◽  
Diploid Nucleus ◽  
C Value ◽  
Salamandra Salamandra ◽  
Cellular Dna ◽  
Pleurodeles Waltl

AbstractThe DNA content per diploid nucleus (2 C-value) was determined for the eight species of Iberian salamanders by flow cytometry. All species showed high values, which are characteristic of Caudata. The species with the lowest value was Pleurodeles waltl (48.25 pg DNA/cell) and Salamandra salamandra had the highest values (70.55 pg DNA/cell). There were no statistical differences between sexes in any of the eight species and DNA content was not directly related with phylogeny. The values obtained are often higher than previously published estimates obtained with older and potentially less accurate methodologies. We discuss the possible relationship between cellular DNA content and reproductive biology. Se determinó el contenido en ADN por núcleo diploide (valor 2-C) por citometría de flujo de ocho especies de salamandras ibéricas. Todas las especies mostraron altos valores, lo que es característico de Caudata. La especie con el menor valor fue Pleurodeles waltl (48,25 pg ADN/célula) mientras Salamandra salamandra tuvo las valores más altos (70,55 pg ADN/célula). No se encontraron diferencias estadísticas entre sexos en ninguna de las 8 especies y el contenido en ADN no se relacionó directamente con la filogenia. Los valores obtenidos son, a menudo, más altos que los publicados previamente con técnicas más antigüas, y potencialmente menos precisas. Discutimos las posibles relaciones entre el contenido en ADN celular y la biología reproductiva de cada especie.

scholarly journals Quantation by flow microfluorometry of total cellular DNA in Acanthamoeba.

1978 ◽  
Vol 26 (9) ◽  
pp. 713-718 ◽  
Author(s):  
P B Coulson ◽  
R Tyndall
Keyword(s):  
Dna Content ◽  
Acanthamoeba Castellanii ◽  
Amoeba Proteus ◽  
Acanthamoeba Polyphaga ◽  
Dna Values ◽  
Flow Microfluorometry ◽  
Identification Of Species ◽  
Acanthamoeba Culbertsoni ◽  
Cellular Dna

The DNA content of five species of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24-48 HR. The trophozoites were harvested, and evaluated for DNA-bound fluorescence. All species tested has DNA values between 2.0-5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.

scholarly journals Nonspecific light loss and intrinsic DNA variation problems associated with feulgen DNA cytophotometry.

1979 ◽  
Vol 27 (10) ◽  
pp. 1377-1379 ◽  
Author(s):  
J P Miksche ◽  
S S Dhillon ◽  
G P Berlyn ◽  
K J Landauer
Keyword(s):  
Nuclear Dna ◽  
Internal Standard ◽  
Root Tip ◽  
Scanning Method ◽  
Dna Variation ◽  
Wavelength Scanning ◽  
Light Loss ◽  
Chicken Erythrocytes ◽  
Dna Measurements

Nonspecific light loss by the cell-wall-plus-cytoplasm (CWC) can cause a 50% increase in Feulgen absorption units in peanut root-tip nuclei as determined by scanning at 450 nm, whereas this phenomenon is not evident with chicken erythrocytes. A two wavelength scanning method of subtracting nonspecific 450 nm absorption from 550 nm Feulgen absorption values eliminated the nonspecific light loss in CWC, However, the two wavelength scanning method is time consuming and somewhat impractical with a regular scanning microdensitometer such as Vickers M85. Elimination of the problem of nonspecific light loss is suggested by careful determination of background setting with the spot position close to the nucleus in CWC. The accuracy of the CWC background setting method was further tested by comparison with subtraction method. The use of plant nucleis as an internal standard in plant DNA measurements was also evaluated. Significant variation among the replicate slides due to the variation in pine nuclear DNA amounts was observed and plant nuclei generally are not reliable internal standards. Mature chicken erythrocytes are recommended as an internal standard because the cell type and metabolic state is known.

Sign in / Sign up

Export Citation Format

Share Document