scholarly journals Energy Transfer between Fluorescein Isothiocyanate and Propidium Iodide – A Problem in the Estimation of Tpotwith the Bromodeoxyuridine–DNA Flow Cytometry Technique?

1999 ◽  
Vol 19 (2) ◽  
pp. 91-98 ◽  
Author(s):  
Maria C. Johansson ◽  
Bo Baldetorp ◽  
Stina M. Oredsson
Keyword(s):  
Flow Cytometry ◽  
Energy Transfer ◽  
Propidium Iodide ◽  
Dna Content ◽  
Fluorescein Isothiocyanate ◽  
Dna Flow Cytometry ◽  
Synthesis Time ◽  
Close Proximity ◽  
Potential Doubling Time ◽  
Potential Doubling

Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 Å) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fullfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine – DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2–3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used.

scholarly journals Quantitation of DNA of Propidium Iodide-stained Nuclei from Ipomoea Species and Sweetpotato Using DNA Flow Cytometry

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 612c-612
Author(s):  
J.R. Bohac
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Ipomoea Batatas ◽  
Colchicine Treatment ◽  
Morning Glory ◽  
Dna Flow Cytometry ◽  
Uv Detector ◽  
Major Barrier ◽  
Ipomoea Species

Sweetpotato, Ipomoea batatas is in the morning glory family, Convolvulaceae, genus Ipomoea, group Batatas. It has many wild Ipomoea relatives that serve as a reservoir of many needed pest and stress-resistance genes. A major barrier to introgression of useful genes is the ploidy gap—sweetpotato is a hexaploid and wild Ipomoeas are diploids and tetraploids. The wild species can be successfully crossed using 2n pollen or by first increasing ploidy by colchicine treatment. The ploidy of such hybrid offpsring can be determined by DNA flow cytometry. My objective was to develop a technique to determine DNA content in Ipomoea and values for DNA content for the major Ipomoea species using the EPIC flow cytometer with a UV detector. Nuclei were extracted and pretreated with cellulase and pectolyase before staining with propidium iodide (PI). A highly linear relationship was found between the DNA content determined by DNA flow cytometry and the ploidy of the closest sweetpotato relatives as determined by chromosome counts. These species were diploid I. trifida, tetraploid I. batatas, and hexaploid I. batatas. DNA content was most similar among other diploid Ipomoea species in the group Batatas and was significantly different in other Ipomoeas not in group Batatas.

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

Diagnosis and risk stratification of Barrett’s dysplasia by flow cytometric DNA analysis of paraffin-embedded tissue

Gut ◽  
2017 ◽  
Vol 67 (7) ◽  
pp. 1229-1238 ◽  
Author(s):  
Won-Tak Choi ◽  
Jia-Huei Tsai ◽  
Peter S Rabinovitch ◽  
Thomas Small ◽  
Danning Huang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Risk Stratification ◽  
Dna Content ◽  
Dna Analysis ◽  
Diagnostic Marker ◽  
Oesophageal Adenocarcinoma ◽  
Dna Flow Cytometry ◽  
Low Grade ◽  
Area Of Interest ◽  
Formalin Fixed Paraffin Embedded

ObjectiveThe diagnosis of dysplasia in Barrett’s oesophagus (BO) can be challenging, and reliable ancillary techniques are not available. This study examines if DNA content abnormality detected by flow cytometry can serve as a diagnostic marker of dysplasia and facilitate risk stratification of low-grade dysplasia (LGD) and indefinite for dysplasia (IND) patients using formalin-fixed paraffin-embedded (FFPE) BO samples with varying degrees of dysplasia.DesignDNA flow cytometry was performed on 80 FFPE BO samples with high-grade dysplasia (HGD), 38 LGD, 21 IND and 14 negative for dysplasia (ND). Three to four 60-micron thick sections were cut from each tissue block, and the area of interest was manually dissected.ResultsDNA content abnormality was identified in 76 HGD (95%), 8 LGD (21.1%), 2 IND (9.5%) and 0 ND samples. As a diagnostic marker of HGD, the estimated sensitivity and specificity of DNA content abnormality were 95% and 85%, respectively. For patients with DNA content abnormality detected at baseline LGD or IND, the univariate HRs for subsequent detection of HGD or oesophageal adenocarcinoma (OAC) were 7.0 and 20.0, respectively (p =<0.001).ConclusionsThis study demonstrates the promise of DNA flow cytometry using FFPE tissue in the diagnosis and risk stratification of dysplasia in BO. The presence of DNA content abnormality correlates with increasing levels of dysplasia, as 95% of HGD samples showed DNA content abnormality. DNA flow cytometry also identifies a subset of patients with LGD and IND who are at higher risk for subsequent detection of HGD or OAC.

scholarly journals Evolution of Genome Size in Duckweeds (Lemnaceae)

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Wenqin Wang ◽  
Randall A. Kerstetter ◽  
Todd P. Michael
Keyword(s):  
Flow Cytometry ◽  
Genome Size ◽  
Propidium Iodide ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Continuous Increase ◽  
Spirodela Polyrhiza ◽  
Wolffia Arrhiza ◽  
Basic Reference

To extensively estimate the DNA content and to provide a basic reference for duckweed genome sequence research, the nuclear DNA content for 115 different accessions of 23 duckweed species was measured by flow cytometry (FCM) stained with propidium iodide as DNA stain. The 1C-value of DNA content in duckweed family varied nearly thirteen-fold, ranging from 150 megabases (Mbp) in Spirodela polyrhiza to 1,881 Mbp in Wolffia arrhiza. There is a continuous increase of DNA content in Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia that parallels a morphological reduction in size. There is a significant intraspecific variation in the genus Lemna. However, no such variation was found in other studied species with multiple accessions of genera Spirodela, Landoltia, Wolffiella, and Wolffia.

scholarly journals NUCLEAR DNA CONTENT IN BLUEBERRY DETERMINED BY FLOW CYTOMETRY

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 580e-580
Author(s):  
Rodomiro Ortiz ◽  
D.E. Costich ◽  
T.P. Meagher ◽  
N. Vorsa
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Haploid Genome ◽  
Dna Flow Cytometry ◽  
Dna Amount ◽  
Ploidy Levels ◽  
Polyploid Evolution ◽  
Haploid Genome Size

DNA flow cytometry was used to determine nuclear DNA content in diploid blueberry species, and 3x, 4x, 5x, and 6x ploidy levels. Relative fluorescence intensity of stained nuclei measured by flow cytometry was a function of the number of chromosome sets (X): Y = 3.7X – 2.3 (r2 = 95.1%). DNA flow cytometry should be useful for ploidy level determination in the seedling stage. A significant linear relationship was established between nuclear DNA content and number of chromosomes (x); DNA (pg) = 0.52 x1 (r2 = 99.8%). Based on this equation the haploid genome DNA amount (1C) was calculated as 0.62 ± 0.08 pg, with an approximate haploid genome size of 602 Mbp/1C. The results indicate that conventional polyploid evolution occured in the section Cyanococcus, genus Vaccinium: the increase in DNA was concurrent with increase in chromosome number. DNA content differences among 2x species were correlated with Nei's genetic distance estimates based on 20 isozyme markers. Most of the variation was among species (49%), with 26% between populations within species, and 25% within populations.

scholarly journals Comparative DNA Flow Cytometric Study of Primary Intraocular and Central Nervous System Lymphomas

2020 ◽  
Vol 8 (2) ◽  
Author(s):  
Teresa Martinu ◽  
Claudia P. Correia ◽  
Andersson M. Figueiredo ◽  
Miguel N. Burnier Jr
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Primary Cns Lymphoma ◽  
Spatial Location ◽  
Intraocular Lymphoma ◽  
Dna Flow Cytometry ◽  
Cns Lymphoma ◽  
Primary Intraocular Lymphoma ◽  
Human Tonsil ◽  
Tissue Samples

Primary intraocular lymphoma is generally considered as a subset of primary CNS lymphoma. This study attempts to show that they may in fact represent distinct entities by comparing their respective proliferation rates using DNA flow cytometry. Four samples of primary intraocular lymphoma and seven samples of primary CNS lymphoma were analyzed, all from paraffin-embedded tissue. All tumors were of the large B-cell type. A normal human tonsil sample was used as a control. Tissue samples were analyzed by DNA flow cytometry, which is a precise and objective method to measure DNA content and cell proliferation of a tumor. S-phase fraction (SPF) and DNA content were measured for each sample. The average SPF for primary intraocular lymphoma was significantly higher than that of primary CNS lymphoma, 23.8 (range: 18.9 to 29.6) versus 15.1 (range: 1.1 to 25.1) respectively. Of the 11 tumors analyzed, 2 brain tumors were aneuploid and 1 eye tumor was peridiploid. All other tumors were diploid. Thus, no significant pattern was detected in the DNA content of the tumors. This lack of clinical significance of tumor aneuploidy is consistent with data reported in the literature. The results of this study indicate that primary intraocular lymphoma is more aggressive and of higher grade than primary CNS lymphoma. The different proliferation rates of intraocular and CNS lymphomas may be explained by either their different spatial location or a distinct genetic composition, the latter reinforcing the hypothesis that the two are fundamentally different entities

Genome size in Arachis duranensis: a critical study

Genome ◽  
2001 ◽  
Vol 44 (5) ◽  
pp. 826-830 ◽  
Author(s):  
Eva M Temsch ◽  
Johann Greilhuber
Keyword(s):  
Flow Cytometry ◽  
Genome Size ◽  
Sea Level ◽  
Dna Content ◽  
Internal Standard ◽  
Critical Study ◽  
Dna Flow Cytometry ◽  
Technical Problems ◽  
Arachis Duranensis ◽  
Collection Sites

Arachis duranensis is a diploid wild relative of the tetraploid cultivated peanut Arachis hypogaea. The literature indicates two 2C genomic DNA mean values (genome size) for A. duranensis, 4.92 and 5.64 pg, and intraspecific variation of up to 11% negatively correlated with altitude above sea level of the collection sites has been reported. Our recent investigations of Arachis species have shown that unrecognized technical problems with peanut material may have influenced previous genome-size data and rendered them open to critical comments. In the present study, 20 accessions of A. duranensis were investigated by means of DNA flow cytometry (propidium iodide staining) and several of these also by Feulgen DNA image analysis. Pisum sativum was used as the internal standard (2C = 8.84 pg). 2C values in A. duranensis were about half those described previously and varied between 2.49 and 2.87 pg (flow cytometry). This variation was statistically significant and reproducible. There was a negative correlation of genome size with latitude and altitude above sea level of the collection sites. Such a correlation had been already found in one of the previous studies. However, the incongruences between the absolute DNA content values obtained in the present investigation and those in the literature point to the importance of carrying out methodological studies on best practice in DNA-content determinations in plants.Key words: Arachis duranensis, genome size, flow cytometry, Feulgen densitometry.

DNA flow cytometry of acinic cell carcinomas of major salivary glands

1990 ◽  
Vol 104 (5) ◽  
pp. 410-416 ◽  
Author(s):  
Adel K. El-Naggar ◽  
J. G. Batsakis ◽  
Mario A. Luna ◽  
Donia McLemore ◽  
R. M. Byers
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Dna Flow Cytometry ◽  
Surgical Removal ◽  
Strong Correlations ◽  
Acinic Cell ◽  
Phase Index ◽  
Number Of Patients ◽  
Ductal Differentiation

AbstractFifteen acinic cell carcinomas from an equal number of patients were analysed for their DNA content and proliferative (S-phase) index by flow cytometry from archival tissues. Seven of the carcinomas manifested a diploid DNA content. None of the patients with diploid acinic cell carcinomas died of their carcinomas and none developed metastases in follow-up periods extending for 10 or more years. Four of eight patients with aneuploid acinic cell carcinomas have died because of their malignancies within a 10 year period after the first surgical removal of the carcinoma. Five of the eight patients exhibited metastases. Although the number of cases does not permit strong correlations between histopathological features, abnormalities in DNA content and outcome of patients, it was noted that carcinomas with prominent necrosis, tubulo ductal differentiation and ‘dedifferentiated’ areas displayed more aggressive biological courses.

scholarly journals DNA flow cytometry of canine mammary tumors: comparative aspects with human breast tumors

2007 ◽  
Vol 59 (5) ◽  
pp. 1163-1168 ◽  
Author(s):  
G.D. Cassali ◽  
A. Salvador ◽  
C. Freitas ◽  
A.P. Dutra ◽  
F.C. Schmitt
Keyword(s):  
Flow Cytometry ◽  
Cyclin D1 ◽  
Dna Content ◽  
Flow Cytometric Analysis ◽  
Mammary Tumors ◽  
Benign Tumors ◽  
Dna Flow Cytometry ◽  
Canine Mammary Tumors ◽  
Significant Difference ◽  
Mib 1

Flow cytometric analysis of DNA content was performed on 28 samples of canine mammary tumors. Nine of them were benign and 19 were malignant. All benign tumors and 11 malignant tumors (57.9%) were diploid (P<0.05). Form the aneuploid tumors, five (26.3%) were hyperdiploid, one (5.3%) hypodiploid, one (5.3%) near triploid and one (5.3%) multiploid. The analysis of the expression of the markers PR and CD31 revealed a significant difference between diploid and aneuploid tumors (P<0.05). The immunoreactivity of PR was higher in diploid tumors, while the immunoreactivity of CD31 was stronger in aneuploid tumors. No difference between the markers MIB-1, c-erbB2, p53 and Cyclin D1 was observed (P>0.05). Using the flow cytometry analysis and immunohistochemistry, it was found a close relationship between aneuploidy and malignant character of neoplasias, progesterone receptor (PR) negative immunostaining and higher microvases density. No correlation between DNA content and S phase or immunoreactivity for the markers MIB-1, p53, c-erbB2 and Cyclin D1 was observed.

scholarly journals Determining Ploidy Level and Nuclear DNA Content in Rubus by Flow Cytometry

2002 ◽  
Vol 127 (5) ◽  
pp. 767-775 ◽  
Author(s):  
Rengong Meng ◽  
Chad Finn
Keyword(s):  
Flow Cytometry ◽  
Pacific Northwest ◽  
Ploidy Level ◽  
Dna Content ◽  
Chromosome Numbers ◽  
Nuclear Dna ◽  
Diploid Species ◽  
Nuclear Dna Content ◽  
Dna Flow Cytometry ◽  
Ploidy Levels

Nuclear DNA flow cytometry was used to differentiate ploidy level and determine nuclear DNA content in Rubus. Nuclei suspensions were prepared from leaf discs of young leaves following published protocols with modifications. DNA was stained with propidium iodide. Measurement of fluorescence of 40 genotypes, whose published ploidy ranged from diploid to dodecaploid, indicated that fluorescence increased with an increase in chromosome number. Ploidy level accounted for 99% of the variation in fluorescence intensity (r2 = 0.99) and variation among ploidy levels was much higher than within ploidy levels. This protocol was used successfully for genotypes representing eight different Rubus subgenera. Rubus ursinus Cham. and Schldl., a native blackberry species in the Pacific Northwest, which has been reported to have 6x, 8x, 9x, 10x, 11x, and 12x forms, was extensively tested. Genotypes of R. ursinus were predominantly 12x, but 6x, 7x, 8x, 9x, 11x, and 13x forms were found as well. Attempts to confirm the 13x estimates with manual counts were unsuccessful. Ploidy level of 103 genotypes in the USDA-ARS breeding program was determined by flow cytometry. Flow cytometry confirmed that genotypes from crosses among 7x and 4x parents had chromosome numbers that must be the result of nonreduced gametes. This technique was effective in differentiating chromosome numbers differing by 1x, but was not able to differentiate aneuploids. Nuclear DNA contents of 21 diploid Rubus species from five subgenera were determined by flow cytometry. Idaeobatus, Chamaebatus, and Anaplobatus were significantly lower in DNA content than those of Rubus and Cylactis. In the Rubus subgenus, R. hispidus and R. canadensis had the lowest DNA content and R. sanctus had the highest DNA content, 0.59 and 0.75 pg, respectively. Idaeobatus had greater variation in DNA content among diploid species than the Rubus subgenus, with the highest being from R. ellipticus (0.69 pg) and lowest from R. illecebrosus (0.47 pg).

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