bluuebunny/crossref_metadata_2025 huggingface dataset:
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

We 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

Sperm 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

In 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

Intracellular 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

Cervical 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

Many 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

Since 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

Quenching 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

Tumor‐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

This 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

When 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

This 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

Fifty‐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

We 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.
Cytophotometric analysis of cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma
Authors: Hanselaar A. G. J. M. , Vooijs G. P. , Van't Hof‐Grootenboer A. E. , Pahlplatz M. M. M.
Published: August 2005

Cytophotometric analysis was performed in nuclei retrieved from paraffinembedded cervical tissue from 57 cases of CIN III. CIN III lesions of patients without invasive squamous cell carcinoma (N=37) were regarded to represent a mixture of progressive and nonprogressive lesions. The CIN III lesions of patients with a synchronous invasive squamous cell carcinoma (N=20) were regarded as representing truly progressive precursor lesions (CIN.INV). Twenty‐one photometric features describing geometrical, density, and texture characteristics were extracted from the digitized nuclear images. Statistical analysis of cytophotometric data indicated significant differences between the group of CIN III lesions and CIN.INV lesions. A cluster analysis, using one co‐occurrency texture feature (S‐HOMOG), one density feature (S‐DI), and two geometrical features (S‐AREA and M‐CIRC), showed that two clusters (C1 and C2) were present in the total group of CIN III and CIN.INV lesions. The vast majority of CIN.INV lesions was member of one and the same cluster C1. The CIN III group appeared to consist of a mixture of two clusters, 54% C1 and 46% C2 lesions. Of patients 45 years or younger, the majority (62%) of CIN III lesions had feature values, corresponding with those of cluster C1, and as such possibly with a potentially progressive course. In patients older than 45 years the percentage of CIN III lesions with C1 feature values was 27%.
Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens
Authors: Hanselaar A. G. J. M. , Vooijs G. P. , Van't Hof‐Grootenboer A. E. , Gemmink J. H. , De Leeuw H. , Pahlplatz M. M. M.
Published: August 2005

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin‐embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin‐Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High‐resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.
Contextual analysis and intermediate cell markers enhance high‐resolution cell image analysis for automated cervical smear diagnosis
Authors: Zahniser D. J. , Wong K. L. , Brenner J. F. , Ball H. G. , Garcia G. L. , Hutchinson M. L.
Published: August 2005

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell‐by‐cell approach can provide.

A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high‐resolution, single cell analysis and the analysis of intermediate cell markers. Thirty‐four samples prepared as mono‐layers and stained with the Feulgen‐Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.
Simultaneous quantification of c‐myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry
Authors: Engelhard Herbert H. , Krupka Jeri L. , Bauer Kenneth D.
Published: August 2005

Variations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a techniaque is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual‐laser (uv and 488 nm) excitation and three fluorescent dyes: FITC, SR 101, and DAPI. FITC‐labelled antibody coupled with indirect immunofluorescence was used ot quantify the c‐myc oncoprotein, whereas SR 101 and DAPI were used ot measure total cellular protein and cellular DNA, respectively.

Flow cytometric measurements of c‐myc oncoprotein were compared to densitometric readings of p64c‐myc SR 101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements corrlated well with those obtained by the biochemical methods.

The usefulness of the technique was further examined following treatment of exponentially growing HL‐60 cells with 2.5 μg/ml cycloheximide for 0 to 12 h. Cycloheximide treatment was found to cause a significant decrease in c‐myc oncoprotein content within 2 h (P<0.05), a relative increase in the proportion of G0/G1 cells and a modest decrease in total cellular protein. This technique appears to provide a rapid, quantitative approach, useful for investigating alterations in cellular growth balance occurring with cell differentiation, neoplastic transformation, or cell treatment with radiation of cytostatic drugs.
DNA distributions in maldescended testes: Hyperdiploid aneuploidy without evidence of germ cell neoplasia
Authors: Clausen O. P. F. , Giwercman A. , Jøsrgensen N. , Bruun E. , Frimodt‐Møller C. , Skakkebæk N. E.
Published: August 2005

Fine‐needle aspiration biopsies and surgical biopsies were obtained from maldescended testes of 149 consecutive men. The aspirates were subjected to quantitative DNA flow cytometry and the surgical biopsy to histological evaluation. From more than 80% of the gonads, sufficient material was obtained for both examinations. A significant hyperdiploid cell population with a mean DNA index of 1.23 (range 1.17–1.31) was found in six gonads. Hyperdiploid aneuploidy was found in gonads without, as well as with, complete spermatogenesis. In none of the six cases did the surgical biopsy show evidence of early testicular neoplasia by morphology or by immunohistochemical methods with antibodies against carcinoma in situ. This indicates that aneuploidies in maldescended testes do not necessarily indicate malignancy. It may be speculated that hyperdiploid aneuploidy is related to the development of preneoplastic lesions.
Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines
Authors: Dooley William C. , Allison David C. , Robertson Joel
Published: August 2005

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non‐modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE‐5, MCa‐11, and HL‐60. The WCHE‐5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa‐11 and HL‐60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa‐11 and HL‐60 M‐ and T‐cell DNA peaks were similar to those of their chromosome number‐peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.
Method for kinetic analysis of drug‐induced cell cycle perturbations
Authors: Ubezio Paolo , Filippeschi Stefania , Spinelli Laura
Published: August 2005

A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0,1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of l‐(2‐chloroethyl)‐l‐nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 μg/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.
Simultaneous use of Rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells
Authors: Srour Edward F. , Leemhuis Tom , Brandt John E. , Vanbesien Koen , Hoffman Ronald
Published: August 2005

The supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin‐HLA‐DR, Texas red‐CD34, allophycocyanin‐CD15, and R123 were fractionated using four‐color immunofluorescence cell sorting. Cells expressing CD34 but not HLA‐DR and CD15 (CD34+ HLA‐DR− CD15−) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R+) or cycling, R123 bright (R++) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34+ HLA‐DR− CD15− R+ cells contained larger numbers of blast cells and colony forming units than CD34+ HLA‐DR− CD15− R++ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.
Flow cytometric analysis of cell cycle‐dependent changes in cell thiol level by combining a new laser dye with hoechst 33342
Authors: Poot Martin , Kavanagh Terrance J. , Kang Hee Chol , Haugland Richard P. , Rabinovitch Peter S.
Published: August 2005

By halogenation of methylfluoresceindiacetate (MFDA) or eosin‐diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)‐excitation system as required by bimane‐based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37°C, and fluorescence is sensitive to pre‐treatment with diethylmaleate but not to buthionine sulfoximine. In a dual‐laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.
Retinoblastoma cell cycling
Authors: Kaleta‐Michaels Shawnya , Char Devron H. , Kroll Stewart M. , Waldman Frederic M.
Published: August 2005

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material.

Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesisphase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48).

DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.
Comparison of autofocus methods for automated microscopy
Authors: Firestone Lawrence , Cook Kitty , Culp Kevin , Talsania Neil , Preston Kendall
Published: August 2005

Traditional autofocus methods were designed for microscopes driven by single processor computers. As computers are developed that exploit massive parallelism when acquiring and analyzing images, parallel cellular logic techniques became available to focus automatically. This paper introduces the reader to both cellular logic techniques for autofocus and a new spectral moment autofocus measure. It then compares these methods with more traditional autofocus methods. It is shown that traditional methods based on measurements of image power‐give the best results when tested on one set of real images and two sets of synthetic images. The next best methods are the cellular logic and spectral moment techniques, while the worst are those based on the image probability density function or histogram.
Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry
Authors: Collins James M. , McLean Grogan W.
Published: August 2005

The transverse location normal to the bilayer surface of a series of n‐(9‐anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 ≥ 3 > 6 ≥ 7 > 9 > 12 > 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.
Pertussis toxin effects on chemoattractant‐induced response heterogeneity in human PMNs utilizing fluo‐3 and flow cytometry
Authors: Omann Geneva M. , Harter Josephine M.
Published: August 2005

Flow cytometric methods were utilized to determine N‐formylpeptide‐induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo‐3. Fluo‐3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo‐3 extrusion was minimal (<10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N‐formyl‐met‐leu‐phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP‐induced cytosolic calcium levels were similar in cells loaded over a 20‐fold range of Fluo‐3‐acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A‐protomer of PT or a very sharp threshold of coupled G‐proteins required to transduce the responses.
Improved FACS‐Gal: Flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs
Authors: Fiering Steven N. , Roederer Mario , Nolan Garry P. , Micklem David R. , Parks David R. , Herzenberg Leonard A.
Published: August 2005

The previously reported FACS‐Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603–2607, 1988) measures E. coli lacZ‐encoded β‐galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, ussing a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence‐activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: (1) use of phenylethy‐lβ‐D‐thiogalactoside, a competitive inhibitor, to inhibit β‐galactosidase activity; (2) reduction of false positives by two‐color measurements; and (3) inhibition of interfering mammalian β‐galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by β‐galactosidase in this assay and the intracellular concentration of β‐galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS‐Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.
An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo‐ and chlorodeoxyuridine incorporated into DNA
Authors: Bakker P. J. M. , Stap J. , Tukker C. J. , van Oven C. H. , Veenhof C. H. N. , Aten J.
Published: August 2005

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat antiBrdUrd monoclonal antibody from Seralab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti‐BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas‐Red conjugated goat antimouse.
Immunofluorescent labeling using covalently linked anti‐phycoerythrin antibodies and phycoerythrin polymers
Authors: Wilson M. R. , Crowley S. , Odgers G. A. , Shaw L.
Published: August 2005

We have developed methods for covalently attaching anti‐phycoerythrin (PE) MAbs to other antibodies, and for using PE or polymers of PE in conjunction with these conjugates to rapidly produce specific, high intensity fluorescent labeling of antigens. The performance of these systems was examined on the surface of microspheres and on the cell surface. The noncovalent means by which PE is incorporated into the label complex in this method makes it possible to use crude algal homogenate successfully as a source of PE in immunofluorescence assays. The intensity of labeling achieved using this method is comparable to, or in some cases better than, that obtained using a direct PE conjugate. With the aim of amplifying the intensity of fluorescence obtained, we produced two types of covalently linked complexes. These were an anti‐PE MAb, designated PE6, linked to itself (i.e., PE6—PE6), and PE linked to itself (i.e., PE—PE). When used in a twostage procedure in place of monomeric PE alone, these complexes increased the intensity of fluorescence obtained on the surface of microspheres by more than fourfold. On the cell surface, the performance of this system varied from one antigen to the next but in most cases was restricted to, at best, a 60% increase in the intensity of fluorescence and in many cases only about a 30% increase.
Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data
Authors: Kamentsky Louis A. , Kamentsky Lee D.
Published: August 2005

We describe a computer‐controlled 10 μm spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above‐threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe‐ based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide‐stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.
Digital morphonuclear analyses of sensitive versus resistant neoplastic cells to vinca‐alkaloid, alkylating, and intercalating drugs
Authors: Pauwels Olivier , Kiss Robert
Published: August 2005

We tested 12 resistant cell lines in vitro in order to evaluate common morphonuclear characteristics induced by various cytotoxic drugs on cell lines of different origins. We used the MXT mouse mammary cancer and the neoplastic J82 and T24 human bladder cell lines, whose variants are either sensitive or resistant to a vinca alkaloid derivative (Navelbine, NVB), to an investigational alkylating agent (PE1001), and to Adriamycin (ADR). We tested cell population variants resistant to NVB + PE1001 + ADR. The level of chemoresistance was evaluated by a colorimetric assay assessing the 50% concentration‐induced inhibition of cellular growth (IC50) brought about by each drug on the growth of each cell variant under study. We show that resistant neoplastic cell nuclei present common morphonuclear characteristics, independent of cell origin (neoplastic mouse mammary versus human bladder cells) and the drug used (vinca alkaloid, alkylating, and intercalating derivatives). Our results further indicate that the phenotype of resistant versus sensitive cells corresponds to cell nuclei populations with smaller nuclei and less nuclear DNA content and, as a consequence, a chromatin texture showing large pale areas with some hyperchromatic clumps.
Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki‐67 or bromodeoxyuridine)
Authors: Larsen Jørgen K. , Christensen Ib Jarle , Christiansen Jette , Mortensen Børge Thing
Published: August 2005

Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)‐stimulated normal human blood lymphocytes for the double staining of (1) Ki‐67 antigen and DNA and (2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd‐labeled cells. With respect to the discrimination between antigen‐positive and ‐negative subpopulations, there was no statistically significant differences between the results from direct (Ki‐67) or indirect, (Ki‐67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.
Simple delay monitor for droplet sorters
Authors: De Grooth Bart G. , Doornbos Richard M. P. , Van Der Werf Kees O. , Greve Jan
Published: August 2005

We have constructed a simple device by which the optimal delay time between optical measurement of a cell and the application of the droplet charging pulse can be determined directly in a flow sorter. The device consists of a stainless steel chamber in which the sorted droplets are collected. In the collection chamber the collected droplets run through a capillary where a continuous fluorescence measurement is made. With a sample of fluorescent particles, the delay time is optimal when the measured fluorescence is maximal. The measuring volume is always filled with the last droplets sorted (about 3,000). With this device, the setting of the delay time can be done in a few seconds without the need for microscopical verification.

The fluorescence in the collection chamber is excited and detected via optical fibers using about 10% of the light of the existing laser from the flow cytometer and an extra photomultiplier.