bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of c‐erbB‐2 protein expression in conjunction with DNA content using multiparameter flow cytometry
Authors: Kelsten Martin L. , Berger Mark S. , Maguire Henry C. , Chianese David A. , Hellman Maria E. , Weiner David B. , Greene Mark I. 
Published: August 2005

AbstractBreast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c‐erbB‐2 protooncogene is amplified in 10–40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c‐erbB‐2 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c‐erbB‐2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c‐erbB‐2 expression in populations of ethanol‐fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c‐erbB‐2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.
Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors
Authors: de L. Davies Catharina , Kovacs Mary 
Published: August 2005

AbstractIn a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl‐lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T‐cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl‐lysine binds to the membrane of heat‐inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl‐lysine signal, the DNA distribution and surface antigen expression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein.
Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a technicon H‐1
Authors: Ross Dennis W. , Bishop Connie , Henderson Amy , Kaplow Leonard 
Published: August 2005

AbstractWe adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H‐1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off‐line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H‐1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.
Quantitative evaluation of radiation‐induced changes in sperm morphology and chromatin distribution
Authors: Aubele M. , Jütting U. , Rodenacker K. , Gais P. , Burger G. , Hacker‐Klom U. 
Published: August 2005

AbstractSperm head cytometry provides a useful assay for the detection of radiation‐induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.
Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors
Authors: Hemmer Joerg 
Published: August 2005

AbstractIn vitro bromodeoxyuridine (BrdUrd) incubated single‐cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.
Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient
Authors: Wang Zhenhua , Chu Guo L. , Hyun William C. , Pershadsingh Harrihar A. , Fulwyler Mack J. , Dewey William C. 
Published: August 2005

AbstractIntracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5‐dimethyl‐2,4‐oxazolidinedione‐2‐14C (14C‐DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF‐loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37°C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0–8.1 agreed‐within 0.1 pH units with that determined by the 14C‐DMO method.There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108‐15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10−3) after heating at 45.5°C either at the normal pHe of 7.4 or at a low pHe of 6.4–6.7.
DNA ploidy and cytophotometric analysis of cervical intraepithelial neoplasia grade III and invasive squamous cell carcinoma
Authors: Hanselaar A. G. J. M. , Vooijs G. P. , Pahlplatz M. M. M. 
Published: August 2005

AbstractCervical intraepithelial neoplasia grade III (CIN III) and squamous cell carcinoma (INV) were examined using DNA ploidy and cytophotometric analysis. Based on hysterectomy, exconisation, and biopsy material from 69 patients in two age categories, analysis was performed in nuclei isolated from selected areas of paraffin‐embedded tissue. High percentages of DNA‐diploidy in INV lesions were found mainly in the group of patients age 45 years or younger. CIN III lesions in women age 46 or older demonstrated high percentages of DNA‐aneuploidy. DNA‐polyploidy was most frequent in CIN III lesions in the younger age category. The results of cytophotometric analysis indicated that the overall mean values of 16 nuclear photometric features discriminated significantly between the whole groups of CIN III (n = 37) and INV (n = 32). On an individual patient level, however, the mean feature values showed a large overlap. Based on the results of a stepwise linear discriminant analysis of patient mean values, a combination of geometrical and run‐length texture features was used to discriminate between CIN III and INV lesions. The correct classification rate was highest in the category of patients in the older age category. The results of this study indicate age related differences in CIN III and invasive squamous cell carcinoma, and they may be of help in assessing cytophotometric features in the study of progressive and non‐progressive CIN lesions.
Triple labeling with two‐color immunoflorescence using one light source: A useful approach for the analysis of cells positive for one label and negative for the other two
Authors: Mansour Issam , Jarraya Mohamed A. , Gane Pierre , Roquin Hélène , Rouger Philippe , Doinel Christian 
Published: August 2005

AbstractMany laboratories do not have access to a flow cytometer allowing three‐color immunofluorescence analysis through the use of multiple light sources. In view of the usefulness of such analyses in the dissection of cell parameters, we describe an approach permitting the study of three labels by using one light source and the two‐color immunofluorescence assay. It is useful for the enumeration of cell subpopulations positive for one label and negative for two or more others as well as for qualitative analysis concerning the expression of these labels. This approach is simple and rapid; it does not require additional material and technical steps other than that used in the two‐color immunofluorescence assay. Briefly, it consists of the use of a label coupled to a dye (PE or FITC or instance) and two different labels coupled to the other dye. An argon ion laser, operating at 488 nm and 60 mW, excites both fluorescein and phycoerythrin conjugated antibodies. We provided a general example, using three hypothetical labels (X, Y, and Z), and four practical applications: CD3+ CD4− CD8− and CD8+ CD16− CD3− peripheral blood lymphocytes, CD2+ CD16− CD3− and CD56+ CD16− CD3− peripheral blood, and decidual infiltrating lymphocytes.
Effect of sonication on paraffin‐embedded tissue preparation for DNA flow cytometry
Authors: Gonchoroff Nick J. , Ryan John J. , Kimlinger Teresa K. , Witzig Thomas E. , Greipp Philip R. , Meyer John S. , Katzmann Jerry A. 
Published: August 2005

AbstractSince the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S‐phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S‐phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S‐phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre‐ and post‐sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.
Quenching of Hoechst 33258 fluorescence in erythroid precursors
Authors: Stokke Trond , Holte Harald , de L. Davies Catharina , Steen Harald B. , Lie Sverre O. 
Published: August 2005

AbstractQuenching of the fluorescence of DNA‐bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoeisis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde‐fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7‐aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (≈10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.
Two‐color flow cytometric analysis of the expression of MAC and MHC Class II antigens on macrophages during tumor growth
Authors: Yurochko Andrew D. , Burger Carol J. , Elgert Klaus D. 
Published: August 2005

AbstractTumor‐bearing host (TBH) macrophages (Mϕ) exhibit immune dysfunction that is concomitant with phenotypic changes. We examined Mϕ subpopulations by changes in the expression of surface antigens Mac‐1, ‐2, ‐3, and Ia on normal and TBH peritoneal and splenic Mϕ. Mϕ were double‐labeled and analyzed by flow cytometry to observe multiple expression of surface antigens. Tumor growth alters the multiple expression of these Mϕ markers. Peritoneal and splenic Mϕ had different Mac+ and Mac+ Ia+ population percentages. In TBH, peritoneal Mϕ had decreased percentages of Mac‐1+2+, Mac‐1+3+, Mac‐2+3+, and Mac+Ia+ Mϕ. This decrease correlated with functional changes in TBH Mϕ. In contrast, there was an increase in Mac‐2−Ia− TBH peritoneal Mϕ. Previously undiscovered Mac‐1+2−3− and Mac‐1−2−3+ populations were found. In contrast to peritoneal Mϕ, there was an increase in the percentage of Mac‐1+2+, Mac‐1+3+, and Mac‐2+3+ splenic TBH Mϕ but, like peritoneal Mϕ, there was a decrease in the percentage of Mac+Ia+ Mϕ. Also, TBH splenic Mϕ showed a smaller but more uniform antigen density than normal host splenic Mϕ. Tumor growth modulated phenotypic alterations in peritoneal and splenic Mϕ subpopulations. Combined with earlier functional studies of Mϕ subpopulations, these data suggested a relationship between changes in Mϕ phenotype and tumor‐induced dysfunction of Mϕ‐modulated immune activity.
The determination of growth rates of individual colonies in agarose using high‐resolution automated image analysisx
Authors: Slocum Harry K. , Malmberg Martin , Greco William R. , Parsons John C. , Rustum Youcef M. 
Published: August 2005

AbstractThis paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation‐modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation‐modifying agents.
Growth of Azotobacter vinelandii with correlation of coulter cell size, flow cytometric parameters, and ultrastructure
Authors: Allman R. , Hann A. C. , Phillips A. P. , Martin K. L. , Lloyd D. 
Published: August 2005

AbstractWhen Azotobacter vinelandii is grown under nitrogen‐fixing conditions, the mean cell volume fluctuates from 2.7 to 6.6 μm3 as determined using a Coulter counter. When NH4Cl is supplied as nitrogen source, the mean cell volume fluctuates from 4.6 to 7.4 μm3. Parallel experiments using flow cytometric measurements show similar characteristic fluctuations in the narrow forward angle light scattering signal and also in cellular protein content as determined using fluorescein isothiocyanate (FITC) fluorescence. Fluctuations in the perpendicular light scatter signal during batch growth are similar for both sets of growth conditions. Changes in cell morphology and ultrastructure are also similar for both sets of growth conditions, as demonstrated by electron microscopic examination. We conclude that narrow forward angle light scatter is a close correlate of cell size, whereas right angle scatter is an indicator of morphological variations other than size.
Simultaneous measurement of DNA content and cell‐surface immunofluorescence of human bone marrow cells using a single laser flow cytometer
Authors: Brons Paul P. T. , Pennings Arie H. M. , Haanen Clemens , Wessels Hans M. C. , Boezeman Jan B. M. 
Published: August 2005

AbstractThis paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell‐surface immunofluorescence (s‐IF). Low density (<1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T‐lymphoid (CD2), B‐lymphoid (CD19), erythroid (anti‐glycophorin‐A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse label was used as second step. Unfixed, MoAb‐labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single‐laser flow cytometer, operating at 488 nm.The effect of the combined staining protocol upon both s‐IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s‐IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S‐phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).Considerable differences in percentages of cells with S‐phase content were seen between the four main bone marrow subpopulations (P<0.0001). The erythroid hematopoietic lineage showed the highest proportion of S‐phase DNA cells (26.4% ± 4.7%), the lymphoid lineage the lowest (2.5% ± 0.8%) while the monocytic lineage (6.1% ± 0.9%) and the myelomonocytic lineage (8.5% ± 1.4%) were in between. Within the lymphoid lineage the percentage of S‐phase cells was significantly lower in T‐cells (2.0% ± 0.7%) compared to B‐cells (5.1% ± 1.5%) (P<0.0001). We conclude that bivariate measurement of S‐phase DNA content and s‐IF is feasible with use of a single laser flow cytometer and that this approach provides important additional biological information. The value of this method to study hematologic malignancies is demonstrated in a bone marrow sample from a patient with erythroleukemia.
Reproducibility of FCM DNA content from replicate paraffin block samples
Authors: Price Joel , Herman Chester J. 
Published: August 2005

AbstractFifty‐five paraffin blocks of morphologically and clinically normal colon specimens were divided in half and the two halves treated as separate aliquots for determination of DNA content using propidium iodide staining and FCM analysis. Forty‐five of the 55 samples showed a peak channel differences between the two aliquots from the same block of > 1 channel. Seven of the 55 specimens showed peak channel difference ≥ 10 channels. Similar results were seen if the mean channel of the G0G1 peak was used in place of the peak channel, with 5 of the 55 specimens showing mean channel differences of ≥ 10 channels. The seven samples with peak channel difference ≥ 10 channels were reprocessed and five of the seven showed reduced peak channel difference on reanalysis. The mean peak channel difference of the 55 samples was 4.3 ± 10.5 (mean ± 2 S.D.) using the first analysis. These data suggest that caution should be used in interpreting ploidy of nuclei extracted from paraffin block material if the interpretation is based on comparing two aliquots from the same block, even if they are processed and analyzed in the same batch.
Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3− human thymocytes
Authors: Zocchi M. Raffaella , Marelli Federica , Poggi Alessandro 
Published: August 2005

AbstractWe describe a method of two‐color immunofluorescence staining which allows the simultaneous analysis of both cytoplasmic antigens and cell entry into the S/G2/M cell cycle phases. This analysis was performed on CD3−‐activated thymocytes obtained from either highly purified CD1−CD3−CD4−CD8− cells or fresh thymus cell suspensions, stimulated with low doses of phorbol‐12 myristate‐13 acetate (0.5 ng/ml) and interleukin‐2. On the 14th day under these culture conditions about 90% of thymocytes did not express CD3 antigen on the cell surface. CD3− cells were further purified by cell sorting, fixed in paraformaldehyde, and permeabilized with Nonidet‐P40. Then these thymocytes were stained by indirect immunofluorescence with monoclonal antibodies identifying T cell‐specific molecules (CD3, CD2, CD28, TCRα/β, and TCR γ/δ) and analyzed for DNA content. Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cell (<80%). Further, the majority of the thymocytes which had entered the S/G2/M phases of the cell cycle (20%) expressed intracellular CD3 and CD28 molecules and reacted with the anti‐β framework βF1 monoclonal antibody. The relationship between the appearance of CD3 and other T cell markers in the cytoplasm, the cell cycle entry, and the thymocyte development is discussed.