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 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.

scholarly journals In irradiation chimeras, K or D regions of the chimeric host, not of the donor lymphocytes, determine immune responsiveness of antiviral cytotoxic T cells

1978 ◽  
Vol 148 (3) ◽  
pp. 805-810 ◽  
Author(s):  
RM Zinkernagel ◽  
A Althage ◽  
S Cooper ◽  
G Callahan ◽  
J Klein
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Cytotoxic T Cells ◽  
Thymic Selection ◽  
Spleen Cells ◽  
Low Responders ◽  
High Responders ◽  
Donor Lymphocytes ◽  
Selection Of

The H-2 haplotype of the chimeric host determines the responder phenotype of maturing T cells. Spleen cells of chimeric mice formed when (K(k) nonresponder to D(b) × K(b) responder to D(b) plus vaccinia)F(1) bone marrow cells were used to reconstitute K(b)D(b) (C57BL/6 D(b) responder) irradiated recipients generated high levels of D(b) plus vaccinia virus-specific cytotoxic T cells. The same stem cells used to reconstitute K(k)D(b) (B10.A (2R) D(b) nonresponder) irradiated recipients resulted in spleen cells that responded well to K plus vaccinia, but responsiveness to D(b) was low. A generally low response to D(k) plus vaccinia, which seems to be regulated by D(k), was confirmed in chimeras. Thus, K(d)D(d) (D(d) plus vaccinia responder) stem cells differentiating in a K(d)D(k) chimeric host failed to generate a measurable response to D(k) plus vaccinia. In contrast, stem cells from K(d)D(k) (D(k) plus vaccinia low responders) differentiating in a K(d)D(d) (K(d) and D(d) high responders to vaccinia) host do generate responsiveness to D(d) plus vaccinia. These results indicate that in chimeras, the Ir phenotype is independent of the donor T cell's Ir genotype, and that thymic selection of a T cell's restriction specificity for a particular H-2 allele of the chimeric host also defines that T cell's Ir phenotype.

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 Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2.

1984 ◽  
Vol 32 (11) ◽  
pp. 1197-1203 ◽  
Author(s):  
D C Allison ◽  
J Meyne ◽  
P F Ridolpho ◽  
K Bose ◽  
M Chakerian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Content ◽  
Nuclear Dna ◽  
Bone Marrow Cells ◽  
Nuclear Dna Content ◽  
Mouse Bone Marrow ◽  
Computer File ◽  
Cellular Dna ◽  
Marrow Cells

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.

Flow cytometry in biodiversity surveys: methods, utility, and constraints

1997 ◽  
Vol 18 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Ilya S. Darevsky ◽  
Robert W. Murphy ◽  
Ross D. MacCulloch ◽  
Cheryl Smith ◽  
Nikolai Orlov ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Liquid Nitrogen ◽  
Dna Content ◽  
Storage Temperature ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Allopatric Species ◽  
Coefficients Of Variation ◽  
Solution Field ◽  
Cellular Dna

AbstractFlow cytometry of blood is a powerful tool for rapidly sorting individual specimens on the basis of cellular DNA content. During biodiversity surveys, the method enabled the early identification of both cryptic sympatric and allopatric species of Vietnamese ranid frogs. This method may be extremely valuable in sorting individuals from other taxa and geographic regions, especially when cellular DNA content is known to vary among closely related taxa, and in tropical situations where crypsis is a relatively common phenomenon. Protocols for preparation of freezing solution, field procedures, preparation of reference standards, and flow cytometric analysis are provided. The best method for field preservation of blood is freezing in liquid nitrogen; field fixation of blood in ethanol was less efficient and resulted in drastically increased coefficients of variation. Once samples have been transferred to freezer storage, they should not be returned to a lower storage temperature in liquid nitrogen.

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

Estimation of food intake in sheep by blood assay for lithium content following ingestion of lithium labelled food

1995 ◽  
Vol 1995 ◽  
pp. 16-16
Author(s):  
J.E. Vipond ◽  
G. Horgan ◽  
D. Anderson
Keyword(s):  
Basal Diet ◽  
Lithium Content ◽  
Biological Mechanisms ◽  
Fixed Amount ◽  
Physiological Factors ◽  
The Past ◽  
Coefficients Of Variation ◽  
Supplementary Feed ◽  
Supplement Intake ◽  
Selection Of

Current rationing systems for sheep and cattle aim to balance a deficit in a basal roughage diet by giving group-fed animals a fixed amount of supplementary food. Assumptions are made that both the intake of basal diet and supplementary feed are average values. Coefficients of variation in individual intake of supplementary feeds of 16-36% have however been observed (Foot and Russel, 1973; Foot et al, 1973) and this variation may be larger (67-107%) where supplements are available as feed blocks (Kendall et al, 1983; Ducker et al, 1981).Recent work on the selection of feed ingredients by sheep (Kyriazakis and Oldham, 1993) and the effect of physiological factors such as parasitism on diet selection (Kyriazakis et al, 1994) suggest that there may be biological mechanisms behind this variation. Estimation of intake of supplements has been difficult in the past, particularly at pasture using chronic oxide and N-alkane indigestible marker systems owing to the need for complete faecal collection procedures and handling procedures that disrupt grazing. A promising new method using lithium as a marker has been developed in Australia (Nolan et al, 1994). This work was undertaken to evaluate the lithium technique for use under UK conditions to elucidate causes of variation in supplement intake of sheep.

Isomer Specific Assay of Ester and Salt Formulations of 2,4-Dichlorophenoxyacetic Acid by High Pressure Liquid Chromatography: Collaborative Study

1978 ◽  
Vol 61 (5) ◽  
pp. 1163-1165 ◽  
Author(s):  
Timothy S Stevens ◽  
Norman E Skelly ◽  
Robert B Grorud
Keyword(s):  
High Pressure ◽  
Collaborative Study ◽  
Internal Standard ◽  
Dichlorophenoxyacetic Acid ◽  
High Pressure Liquid Chromatographic ◽  
Coefficients Of Variation ◽  
Standard Solution ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Liquid Chromatographic

Abstract A high pressure liquid chromatographic (HPLC) assay of ester and salt formulations of 2,4-D has been collaboratively studied. The method is specific for 2,4-D isomer and resolves all known impurities from 2,4-D and the internal standard p-bromophenol. In situ saponification, at room temperature, is performed by adding a combined saponification-internal standard solution to ester products. The same saponification- internal standard solution is added to amine salts and the analytical standard. The injected aqueous potassium salt solution of 2,4-D is then converted to the acid form by an acidic buffered mobile solvent of 20% acetonitrile in water. Optimum chromatography is attained by a mobile solvent pH of 2.95 in a reverse phase microparticulate column, by ion suppression. Each of the 9 collaborators received 3 different ester and 2 different amine formulations of 2,4-D. The coefficients of variation of 2,4-D acid equivalent ranged from 1.22 to 1.59%. The method has been adopted as official first action.

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