bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia
Authors: Arkesteijn G. J. A. , Erpelinck S. L. A. , Martens A. C. M. , Hagenbeek A.
Published: June 2005

Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde‐fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin‐and DIG labeled chromosome specific centromere probe. A number of probes were tested in the suspension hybridizations. The method yielded fluorescent hybridization signals that allow discrimination between Y chromosome positive and negative nuclei when analyzed by flow cytometry. The method is especially suited for analysis of bone marrow cells derived from patients who have received a sex‐mismatched allogeneic bone marrow transplantation. Male leukemia cells with a trisomy for chromosome 8 were mixed with normal female cells and simultaneously hybridized in suspension with a DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120‐fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000. © 1995 Wiley‐Liss, Inc.
Cell volume and shape oscillations in rat type‐II somatotrophs at hypotonic conditions
Authors: Engström Karl Gunnar , Sävendahl Lars
Published: June 2005

The size and shape of growth hormone (GH)‐producing rat type‐II somatotrophs was studied during osmotic manipulation. When somatotrophs were exposed to large osmotic stress (200 and 225 mOsm), the peak projected cell area (PCA) was 132.9% ± 12.6% and 116.8% ± 2.8% (P < 0.01) and triggered a regulatory volume decrease (RVD) to avoid lysis. At lower osmotic stress (250 mOsm), the rate of swelling was slower, and the volume reached a steady state at 109.4% ± 2.4% (P < 0.05) and was without RVD. At 275 and 287 mOsm, the swelling was delayed [PCA peak at 3–4 min; 105.8% ± 1.5% (P < 0.05) and 104.2% ± 1.7%] and then showed repeated synchronized cycles of swelling and shrinkage (P < 0.05). The data suggest that somatotrophs may have more than one mechanism for volume regulation. One mechanism is for large swelling (classic RVD response), whereas the other represents more physiological mechanisms for regulating cell volume within a more limited geometry range. For low osmotic stress (250–287 mOsm), the somatotrophs became less spherical during swelling and, thus, were without membrane dilation. Therefore, this type of volume regulation must work independently from membrane stress. Related volume regulation mechanisms may underlie the previously observed volume fluctuations in somatotrophs seen during secretory stimulation with GH‐releasing hormone. © 1995 Wiley‐Liss, Inc.
Anti‐CD3‐Induced Apoptosis in T‐Cells From Young and Old Mice
Authors: Chrest Francis J. , Buchholz Meredith A. , Kim Young Ho , Kwon Taeg‐Kyu , Nordin Albert A.
Published: June 2005

Light scatter measurements using flow cytometry indicated that T cells from young and old mice undergo apoptosis following activation with immobilized anti‐CD3. The percentage of cells in apoptosis after 20 h activation was significantly greater (p<.001) in cultures containing cells from older animals. The mean percentages of apoptotic T cells from young and old mice after 20 h activation were 19.3% and 33.0%, respectively. The proportion of viable cells after 20 h activation was significantly higher (p<.003) in the young (mean=78.4%) than in the old animals (mean =: 65.8%). Simultaneous measurements of light scatter and fluorescence indicated that apoptotic T cells contained both the CD4+ and the CD8 + T cell phenotypes. The frequency of apoptotic CD8+ T cells was elevated (p<.007) in older animals, where the mean percentage was 15.1%, compared to 5.3% in the young. The most dramatic difference between young and old (p<.0008) was seen in the percentages of viable CD4+ T cells after 20 h activation. The mean viable CD4+ T‐cell percentage was 33.7% in the young and 21.4% in the old. CD4+ cells expressing high levels of CD45RB (CD45RBhi) after activation for 20 h possessed light scatter and bright fluorescence properties characteristic of viable cells, whereas CD4+/CD45RB10 density cells could be identified as apoptotic based on their decreased CD4 fluorescence and scatter characteristics. CD4+ cells from young animals were predominantly CD45RBhi, whereas CD4+ cells from the old had greater levels of CD454RB10 cells.

In addition to light scatter changes, measurement of DNA content after 40 h activation revealed the presence of a sub‐G1 DNA apoptotic peak and a viable cell cycle distribution. After 40 h of activation, there was an increase in the percentage of apoptotic cells in both young and old mice, with the greatest increase seen in the cells from older animals. Further evidence supporting the process of apoptosis in 40 h‐activated cells was confirmed by the appearance of DNA strand breaks detected by in situ nick translation. © 1995 Wiley‐Liss, Inc.
A model for evaluation of in situ hybridization spot‐count distributions in tissue sections
Authors: Pahlplatz M. M. M. , de Wilde P. C. M. , Poddighe P. , v. Dekken H. , Vooijs G. P. , Hanselaar A. G. J. M.
Published: June 2005

The interpretation of in situ hybridization (ISH) spot‐count distributions, obtained from evaluation of ISH signals in tissue sections, is complicated by the unknown impact of nuclear truncation and of the localization of ISH spots within the nuclei. In this study, a mathematical model was developed to investigate the effects of nuclear truncation and of the distribution of ISH spots within the nucleus on the ISH spot‐count distribution in tissue sections. In this model, it was assumed that nuclei are spherical and of constant diameter and that ISH spots have negligible size and are distributed randomly within the nucleus (“volume model”) or along the nuclear membrane (“surface model”). A minimal nuclear profile diameter was introduced in order to study the effect of rejecting small nuclear fragments for spot‐count evaluation. Given the section thickness, the nuclear size, the minimal nuclear profile diameter, and the true number of ISH spots per nucleus and their spatial distribution within the nucleus, the model predicts the proportion of nuclei observable in the section with a specific number of ISH spots. A program that performs the model calculations was developed for PC and is available upon request.

For section thickness greater than 50% of the nuclear diameter, the main effect of increasing section thickness on spot‐count distributions was the increase of the proportion of nuclei with the true chromosome copy number of spots. For lower section thickness, the total distribution shifted towards lower spot frequencies. The influence of the minimal profile diameter was most notable for values close to the nuclear diameter. The effect of the localization of ISH spots within the nucleus was shown to be prominent, especially for sections with thickness smaller than the nuclear diameter. Good correspondence between model‐predicted distributions and measured distributions was obtained using the volume model and taking into account only large nuclear profiles. © 1995 Wiley‐Liss, Inc.
Dyes providing increased sensitivity in flow‐cytometric dye‐efflux assays for multidrug resistance
Authors: Frey Tom , Yue Stephen , Haugland Richard P.
Published: June 2005

In an effort to improve detection of P‐glycoprotein‐mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays. Two dyes (SY‐38 and SY‐3150) were found that provided better sensitivity. These dyes were effluxed by a cell line known to be mdr‐positive (P388/R84) but not by an mdr‐negative cell line (P388). Efflux was blocked by both verapamil and cyclosporine A. Efflux from KG1a cells was less than from P388/R84, just as has been seen with R123 and daunomycin. In further experiments, a model system was used to demonstrate two‐color immunofluorescence plus efflux measurements. This was done using a sequential staining method. A procedure was devised that, at least for this model system, allowed single‐step staining with both dye and antibody. The sensitivity of the efflux measurement was slightly compromised by using this one‐step staining method. © 1995 Wiley‐Liss, Inc.
Occurrence and a possible mechanism of penetration of natural killer cells into k562 target cells during the cytotoxic interaction
Authors: Radoškevića Katarina , van Leeuwen Anne Marie T. , Segers‐Nolten Ine M. J. , Figdor Carl G. , de Grooth Bart G. , Greve Jan
Published: June 2005

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo)emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in‐conjugation). Virtually all in‐conjugated target cells exhibited polymerized actin. Killer cells of in‐conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in‐conjugation were observed. A kinetic study showed that in‐conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in‐conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in‐conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in‐conjugation.

Our study demonstrates that in‐conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism. © 1995 Wiley‐Liss, Inc.
High affinity binding of 7‐aminoactinomycin D and 4′,6‐diamidino‐2‐phenylindole to human neutrophilic granulocytes and lymphocytes
Authors: Loborg Helena , Lindén Eva , Lönn Anita , Skoglund Per , Rundquist Ingemar
Published: June 2005

The binding behavior of the DNA binding dyes 7‐aminoactinomycin D (7‐AAMD) and 4′,6‐diamidino‐2‐phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7‐AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome‐free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7‐AAMD binding sites remained in the unextracted PFA‐fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7‐AAMD compared to lymphocytes. DAPI had a much higher affinity than 7‐AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ. © 1995 Wiley‐Liss, Inc.
Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in p‐glycoprotein‐expressing MCF7 human breast adenocarcinoma cells
Authors: Merlin Jean‐Louis , Marchal Sophie , Ramacci Carole , Dieterlen Alain , Schultz Guy , Lucas Catherine , Poullain Marie‐Gwenaelle , Berlion Maryse
Published: June 2005

A triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human brest adenocarcinoma MCF7DXR cell line expressing P‐glycoprotein. In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis. Compared to verapamil and cyclosporin A at 2 and 5 μol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells. Significant activity of S9788 was observed at 2 μol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line. Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (>90%) at 2 μol/liter, whereas cyclosporin A reached this level of activity at 5 μol/liter, and verapamil was always less active at both concentrations. These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action. © 1995 Wiley‐Liss, Inc.
Flow cytometric study of differentiating cultures of Bacillus subtilis
Authors: Chung John D. , Conner Stewart , Stephanopoulos Gregory
Published: June 2005

We report on (1) the development of aflow cytometry‐based technique for detecting β‐galactosidase in differentiating cultures of Bacillus subtilis and (2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5‐octanolyaminofluorescein di‐β‐D‐galactopyranoside (C8‐FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8‐FDG staning mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8‐fluorescein and PKH26 allow nonspecific C8‐FDG staining in this control population to be monitored using two‐color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulationg genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild‐type and mutant cultures early during the development process of spore formation. © 1995 Wiley‐Liss, Inc.
Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates
Authors: Ganesh S. , Klingel S. , Kahle H. , Valet G.
Published: June 2005

Aminopeptidases (AP) are ubiquitously occuring, nonspecific exopeptidases involved in protein degradation. They cleave the N‐terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of AP (EC 3.4.11.X) activity in viable cells by flow cytometry was the subject of this study because of its general biological and clinical interest. Different bis‐substituted rhodamine 110 (R110) peptide derivatives were synthesised and used as AP‐ and exopeptidase (EC 3.4.13.X–EC 3.4.14.X) substrates for flow cytometric measurements. Intracellular AP activities in viable lympho‐, mono‐, granulo‐, and thrombocytes were detected by fluorescence increase from R110 following intracellular substrate cleavage. Eukaryotic‐AP do not cleave D‐aminoacids and hence NH2(D‐Leu)2R110 substrate served as negative control. Specific substrate cleavage by AP is shown by complete inhibition of fluorescence generation following preincubation of cells with leucinechloromethylketone inhibitor. R110 AP‐ and exopeptidase substrates are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracellular fluorescence. © 1995 Wiley‐Liss, Inc.
Evaluation of red blood cell lysing solutions for the detection of intracellular antigens by flow cytometry
Authors: Tiirikainen Maarit I.
Published: June 2005

When analyzing leukocyte cell surface antigens by flow cytometry, leukocytes are usually first labeled in whole blood and the red blood cells are finally lysed with lysing solutions. The erythrocytes are lysed, but the leukocytes are expected to remain intact. Six commercial red blood cell lysing methods were investigated for possible leukocyte permeabilization effect. The effectiveness of permeabilization was studied by propidium iodide staining, and the detectability of intracellular antigens was studied by using monoclonal antibodies toward two model antigens. Most of the lysing methods caused permeabilization of at least part of the leukocytes, but only one method, already found in our previous studies, was applicable for complete permeabilization of leukocytes and for detection of intracellular antigens alone or simultaneously with the cell surface antigens. © 1995 Wiley‐Liss, Inc.
Evaluation of T‐lymphocyte subsets present in semen and peripheral blood of healthy donors: A report from the heterosexual transmission study
Authors: Denny Thomas N. , Scolpino Anthony , Garcia Ambrosia , Polyak Andrew , Weiss Stanley N. , Skurnick Joan H. , Passannante Marian R. , Colon Jose
Published: June 2005

The purpose of this study was to accurately determine the T‐lymphocyte subsets found in semen from health volunteers, to evaluate the impact of repeated ejaculation on the frequency or type of immune cells present in semen, and to compare subset analysis in semen to that in the peripheral blood. To accomplish this, a flow cytometric method was developed to identify and count immunophenotypically distinct cells present in semen. Fresh semen samples and peripheral blood were collected over three consecutive days from nine healthy donors. Donors had normal ejaculate volume, sperm count, sperm motility, morphology, and leukocyte count. No significant intra‐donor differences were seen in these parameters over time. No significant differences were observed in the percentage of CD3+ cells, CD4+ cells, CD8+ cells, and the CD4:CD8 ratio in semen on consecutive days. However, within the CD4+ subset, when naive and memory CD4+ cells were measured, some day to day variability was suggested. No significant differences in CD3+, CD4+, CD8+, CD4/CD8 ratio, or naive and memory subsets were seen in the peripheral blood between sampling days. When semen was compared to peripheral blood some differences in immune subset values were observed, with an increase in the percentage of memory CD4+ cells in semen being the most striking. This finding may be relevant to HIV transmission, since others have shown that this cell may be preferentially infected with HIV and is the primary reservoir for virus in infected individuals. © 1995 Wiley‐Liss, Inc.
Autofluorescence correction for fluorescence in situ hybridization
Authors: Szöllösi János , Lockett Stephen J. , Balázs Margit , Waldman Frederic M.
Published: June 2005

Optimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus‐specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel‐by‐pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image‐processing approach to autofluorescence correction may widen the applicability of gene‐specific probes in FISH analysis of tumor material. © 1995 Wiley‐Liss, Inc.
Genetic and phenotypic heterogeneity of human malignancies: Finding order in chaos
Authors: Shackney Stanley E. , Shankey T. Vincent
Published: June 2005

The presence of cellular heterogeneity within human tumors has been recognized for many years. Current concepts regarding the clonal origin of human neoplasms, and recent advances in the study of successive genetic changes that occur during tumor evolution may now make it possible to understand in greater depth the biological and clinical implications of intra‐tumor heterogeneity at both the phenotypic and genotypic levels. In order to explore these concepts further, and to better identify the potential contributions that flow and image cytometry can make to our understanding of tumor heterogeneity, a session of the 1994 ISAC Congress was dedicated to plenary presentations on human can cell heterogeneity. Here, we provide a brief overview of the genetic evolutionary progression of human cancers, some considerations of clinically important phenotypic and genotypic markers, and an outline that might serve as a basis for framing relevant issues that are ammenable to further study.

All Nature is but Art, unknown to thee;

All Chance, Direction, which thou canst not see;

All Discord, Harmony not understood:

All partial Evil, universal Good.

(Alexander Pope, Essay on Man, end of Epistle 1) © 1995 Wiley‐Liss, Inc.
Geographic distribution of co‐dominant DNA stemlines in breast carcinoma
Authors: Visscher D. , Dolan P. , Ottosen S. , Crissman J.
Published: June 2005

Breast carcinomas often contain multiple DNA stemlines in flow cytometric DNA histograms. However, due to mixing during tissue disaggregation the microanatomical relationship between the cells which comprise distinct stemlines is unclear. We performed image cytophotometric DNA analysis (IA) on two separate areas of intact tissue sections of 19 breast carcinomas which were selected on the basis of flow cytometric (FCM) DNA content heterogeneity (i.e., multiple stemlines). For comparison, similar analyses were performed on seven tumors with unimodal FCM DNA histograms. Six of the 7 tumors (86%) with unimodal FCM histograms were also unimodal in both IA DNA histograms. Among tumor with heterogeneous FCM DNA histograms, the presence of multiple stemlines was confirmed in IA DNA histograms in 16/19. In nine of these 16 cases, multiple DNA stemlines having similar DNA indices were present in both areas of neoplasm examined with IA. The remaining seven cases displayed unimodal IA histograms in both areas, however DNA indices differed between the two histograms. These findings imply that cell populations corresponding to flow cytometrically detected DNA stemlines are often intimately admixed, even within geographically separated portions of breast tumors. This pattern suggests that productive interactions between genetically distinct tumor populations may lead to stable co‐dominance of ancestral clones during progression of some breast carcinomas.
Assessing sequential oncogene amplification in human breast cancer
Authors: Janocko Laura E. , Lucke Joseph F. , Groft David W. , Brown Kathryn A. , Smith Charles A. , Pollice Agnese A. , Singh Sarita G. , Yakulis Robert , Hartsock Robert J. , Shackney Stanley E.
Published: June 2005

Studies of amplification and/or overexpression of c‐myc, HER‐2/neu, and H‐ras in breast cancer have shown that each is associated with a poor prognosis. The purpose of this study was to explore the possibility that there is a preferred sequence of amplification of these oncogenes in breast cancer. The frequencies of amplification and patterns of co‐amplification of c‐myc, HER‐2/neu, and H‐ras were studied in a group of 84 breast cancers. The data suggested a preferred sequence of amplification that consisted of c‐myc amplification‐HER‐2/neu amplification‐H‐ras amplification. This model was supported by loglinear analysis. In addition, the levels of amplification of JC‐A, a DNA fragment newly isolated from a patient with advanced breast cancer, were studied in 61 of these cases. The data suggested that JC‐A amplification occurred early. Loglinear analysis supported a model in which JC‐A amplification occurred either before or after c‐myc amplification but was unrelated to Her‐2/neu or ras amplification. © 1995 Wiley‐Liss, Inc.
DNA Ploidy and proliferation heterogeneity in human prostate cancers
Authors: Shankey T. Vincent , Jin Jia‐Kuan , Dougherty Scott , Flanigan Robert C. , Graham Sharon , Pyle Joseph M. , Graham Sharon
Published: June 2005

DNA ploidy determinations have been shown to have clinical application in predicting disease progression, survival, or response to anti‐androgen therapies in prostate carcinomas. Since intra‐tumor heterogeneity may have a profound effect on DNA measurements, we determined the frequency of DNA ploidy and proliferation (here S‐phase fraction) heterogeneity in early prostatic carcinomas, and estimated the potential impact of heterogeneity on predicting disease course, survival, or response to therapy.

Using image and flow cytometric analysis of archival, paraffin‐embedded prostate tumors, we measured DNA ploidy in individual foci of prostatic carcinoma in stage T1a, T1b and T1c disease. Image analysis studies included the use of Feulgen stained tissue sections, and a comparison of these results with flow cytometric DNA ploidy determinations on nuclei isolated from the same tumor foci. Flow cytometry was also used to measure DNA Index and tumor S‐phase fraction, in some cases using multiparameter analysis of isolated nuclei to determine DNA content and the level of the proliferation‐associated antigen, p105.

Our results indicate that DNA aneuploid foci of prostate carcinoma are infrequently seen in stage T1a disease (13% of the individuals studied), and that the presence of both DNA diplold and aneuploid foci in the same sample is seen in less than 10% of these individuals. Stage T1b and T1c tumors containing only DNA diploid nuclei are seen, though these are likely most common in low volume, low Gleason grade tumors. By using flow cytometry to compare these results with those using image analysis of the same tumor foci, we demonstrated that the majority (>75%) of these aneuploid tumors are DNA tetraploid. Our data on prostate tumor S‐phase fractions indicate that DNA diploid tumors generally have a lower S‐phase than DNA aneupoloid foci (including comparisons of DNA diploid and aneuploid foci in the same prostate tumor). These results support the model that early prostate tumors are DNA diploid and have a low S‐phase, and that these tumors likely evolve to DNA tetraploid tumors with a similar low S‐phase fraction. © 1995 Wiley‐Liss, Inc.
Heterogeneity of anthracycline retention and response to efflux blockers in human tumors
Authors: Krishan Awtar
Published: June 2005

Rapid cellular efflux of certain natural products used in cancer chemotherapy leads to reduced cytotoxicity and resistance. Multiple Drug Resistance (MDR) gene related glycoproteins are believed to act as the drug efflux pump. Several non‐cancer therapy drugs (e.g., verapamil, phenothiazines) compete for the p‐glycoprotein pump and thus can block efflux of a chemotherapeutic agent and overcome cellular resistance. Anthracyclines, such as adriamycin, are intrinsically fluorescent and thus their cellular retention can be determined by laser flow cytometry. This method has been used to study heterogeneity in cellular retention of anthracyclines and response to efflux blockers in human tumor cells. These studies have led to generation of clinical protocols where laser flow cytometry is used to monitor and overcome heterogeneity in drug retention of tumor cells from patients treated with a combination of adriamycin and efflux blockers. © 1995 Wiley‐Liss, Inc.
Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis
Authors: Visscher Daniel W. , Wallis Tracie , Ritchie Carole A.
Published: June 2005

We compared flow‐cytometric DNA histogram pattern to counts of 4 fluorescent‐labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from the corresponding formalin‐fixed deparaffinized thin tissue section (TS) in 25 breast lesions (9 invasive cardqomas, I duct carcinoma‐in‐situ, 5 fibroadenomas, 10 fibrocystic change). In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0–43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50–150% greater than in matched WN preparations (chromosome 1–21.7%, chromosome 7–21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in WN compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometrnic DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10–25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples.

We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability.
Near‐IR dyes in three‐color volumetric capillary cytometry: Cell analysis with 633‐ and 785‐nm laser excitation
Authors: Lee Linda G. , Woo Sam L. , Head David F. , Dubrow Robert S. , Baer Thomas M.
Published: June 2005

Several fluorescent dyes that absorb in the nearinfrared are described. The photostability and aggregation properties of the dyes were examined. Two of the dyes, BHMP and BHDMAP, emit at 805 nm and were useful dyes for protein labeling. A dual‐laser, three‐color scanning instrument was constructed. CD3 + CD4 + and CD3 + CD8 + populations were enumerated in undiluted, whole blood based on the fluorescence of Cys5, Cy5.5 and BHMP. © 1995 Wiley‐Liss, Inc.
Spatial dependence of the optical collection efficiency in flow cytometry
Authors: Goodwin Peter M. , Ambrose W. Patrick , Martin John C. , Keller Richard A.
Published: June 2005

The sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [∼1] picoliter (p1) sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurements are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed. © 1995 Wiley‐Liss, Inc.
Cell membrane labeling with fluorescent dyes for the demonstration of cytokine‐induced fusion between monocytes and tumor cells
Authors: Spötl Ludwig , Sarti Alessandra , Dierich Manfred P. , Möst Johannes
Published: June 2005

Fusion between monocytes and tumor cells has been suggested as a cause for tumor metastasis. The aim of the present study was to establish an in vitro fusion model representing the in vivo situation as close as possible. For this purpose fusion between cells was induced by cytokine containing conditioned medium. In order to prove that hybrid formation between tumor cells and monocytes occurs, a two‐color‐fusion‐assay based on membrane labeling with the fluorochromes PKH 2 (green) and PKH 26 (red) was established. These fusion experiments were analyzed by microscopy and, in addition, by flow cytometry. The attempt to induce fusion between monocytes and several tumor cell lines of hematopoietic origin revealed quite diverse results. The most extensive hybrid formations were seen with TALL, a T‐lymphocytic tumor line. The monocytic tumor line HL60 and the B‐lymphocytic tumor line BL41 also clearly yielded hybrids with monocytes but in smaller numbers. With some other hematopoietic tumor lines no evidence for hybrid formation was detected. These studies indicate that fusion of normal monocytes with certain tumor cells may be induced under conditions that may occur in comparable manner in vivo. © 1995 Wiley‐Liss, Inc.
Heterogeneous calcium flux in peripheral T cell subsets revealed by five‐color flow cytometry using log‐ratio circuitry
Authors: Roederer M. , Bigos M. , Nozaki T. , Stovel R. T. , Parks D. R. , Herzenberg L. A.
Published: June 2005

Calcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence paramein. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets. © 1995 Wiley‐Liss, Inc.
Flow cytometric immunofluorescence and DNA analysis: Using a 1.5 mW helium‐neon laser (544 nm)
Authors: Hudson James C. , Porcelli Ronald T. , Russell Thomas R.
Published: June 2005

We evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76 μm Sortsense flow cell. The two applications chosen were immunofluorescence and DNA analysis. We measured the fluorescence threshhold of phycoerytherin calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Argon laser. In addition, a three‐color configuration yielded excellent results. Cell analysis of CD4PE, CD3ECD and CD19Cy‐Chrome with the HeNe laser and Argon laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full CV values for whole blood DNA fluorescence using the green laser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluorescence. The wavelength of the 544 nm laser is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and Cy‐Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantages of this laser for the research laboratory are small size, no cooling fan, low power requiremements and low cost. © 1995 Wiley‐Liss, Inc.
A rapid mix flow cytometer with subsecond kinetic resolution
Authors: Nolan John P. , Posner Richard G. , Martin John C. , Habbersett Robert , Sklar Larry A.
Published: June 2005

Kinetic approaches are valuable tools for mechanistic studies of cell function. Flow cytometry is well suited to make sensitive kinetic measurements, but the time required to deliver mixed samples to the point of measurement (10–20 s in a conventional cytometer) limits analysis of rapidly occurring events. To address this limitation, we adapted a syringe‐based stopped‐flow rapid mixing device to a modified commercial flow cytometer to achieve mixing and measurement of sample in under 1 s. Because such screw‐driven mixers are designed to deliver fluid at rates of microliters per millisecond and cytometers accept samples at microliters per second, the syringe mixer was modified with a screw to allow sample delivery at rates as low as 1.8 μ1/s. A custom‐made nozzle holder featuring a fast‐acting three‐way sample delivery valve and a 1.5‐ μ1 dead volume was designed for a Becton Dickinson FACS stream‐in‐air flow nozzle. Syringe motors and valves are computer controlled, as is the start signal for an adjustable time ramp. A stable sample stream can be established within the sheath stream in less than 1 s, enabling fluorescence measurements of microspheres with coefficients of variation of ∼5%. Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at times of under 300 ms. Efficient mixing of reagents is demonstrated by the iodide quenching of microspheres surface labeled with fluorescein isothiocyanate (FITC). The instrument is capable of quantitatively proportioning cells and reagent, thereby allowing precise control of reagent concentration and dilution. Rapid kinetic measurements of intact cells are demonstrated by FITC‐formyl peptide binding to cell surface receptors. © 1995 Wiley‐Liss, Inc.
Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance
Authors: Sukhorukov Vladimir L. , Djuzenova Cholpon S. , Frank Hermann , Arnold W. Michael , Zimmermann Ulrich
Published: June 2005

Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0‐Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5–3.0 kV/cm, 40 μs) in medium containing 25–50 μg/ml PI at 21–23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: (1) intact living cells with CF but without PI (these form >95% of the prepulsed population), (2) transiently electropermeabilized but resealed cells showing both CF and low‐level PI fluorescence, and (3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic “pulse medium,” the membranes resealed after electropulsing with a time constant (τR) of about 2 min. In 150 mOsm medium, resealing was faster (typically τR ∼ 0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse‐induced voltage (CVradius ∼ 10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole‐cell capacitance (Cc), was taken into account. Evaluation of the Cc values with single‐cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. © 1995 Wiley‐Liss, Inc.
Effect of cell density on the expression of adhesion molecules and modulation by cytokines
Authors: Stanley A. J. , Banks R. E. , Southgate J. , Selby P. J.
Published: June 2005

Little is known about the regulation of integrin expression. Using flow cytometry, we have characterized the expression of integrin subunits and other adhesion molecules in bladder and colonic cell lines and studied their modulation by the cytokines tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). We show that although TNF‐α and IFN‐γ may modulate integrin expression, these effects are, in the case of some integrin subunits, secondary to cytokine‐induced changes in cell density. Cell density‐dependent changes in the expression of α2‐, α3‐, α6‐, β1‐, and β4‐integrin subunits were seen. IFN‐γ produced density‐independent decreases in expression of α2‐ and β1‐integrins and increases in expression of intercellular adhesion molecule‐1 (ICAM‐1), α3‐, α6‐, and β4‐integrins. TNF‐α produced increases in β1‐integrin and ICAM‐1 expression which were also density‐independent. The expression of some integrin subunits is clearly density‐dependent and the alterations seen are dependent on the cell line studied. This study illustrates the importance of including appropriate density‐related controls in all investigations examining adhesion molecule expression. © 1995 Wiley‐Liss, Inc.
Rapid detection of antimicrobial activity using flow cytometry
Authors: Durodie J. , Coleman K. , Simpson I. N. , Loughborough S. H. , Winstanley D. W.
Published: June 2005

Different antibiotic classes vary considerably in their modes of action and hence in their effects on the bacterial cell. Flow cytometry was used to analyse E. coli cells treated with five antibiotics differing a their modes of action. The ratio of protein content, as measured by fluorescence, to forward light scatter (i.e., FL1:FSC) provided a simple and reliable way of detecting within 2 h of treatment antimicrobial activity at a 0.5 minimum inhibitory concentration. This ability to detect antimicrobial activity rapidly offers considerable advantages in drug research for the rapid detection of novel antimicrobials and may, with further development, find a use in the clinic for rapid susceptibility testing as an aid to the selection of therapy. © 1995 Wiley‐Liss, Inc.
DNA aneuploidy in acute myeloblastic leukemia is associated with a high expression of lymphoid markers
Authors: Vidriales M. B. , Orfao A. , López‐Berges M. C. , González M. , López‐Macedo A. , Ciudad J. , López A. , Cañizo M. C. , Garcia M. A. , Miguel J. F. San
Published: June 2005

In the present study the DNA cell content of 205 de novo acute myeloblastic leukemia (AML) patients is analyzed at flow cytometry in order to determine both the incidence of DNA aneuploidy in AML patients and the clinical and biological characteristics of AML aneuploid cases. All technical procedures were performed in accordance with the proposed guidelines of the DNA Cytometric Consensus Conference. Our results show that the incidence of DNA aneuploidy is quite low (4.8%), with most cases (n = 8) hyperdiploid and only a small proportion (n = 2) hypodiploid. No major differences were detected between the aneuploid and diploid cases with respect to the clinical and prognostic disease characteristics. Regarding the immunophenotype of the blast cells, the aneuploid cases displayed both a higher incidence of immature myeloblastic phenotype and a greater expression of lymphoid‐associated antigens. In summary, our results show that AML patients display a quite low incidence of DNA aneuploidy and that despite the fact that these cases do not display particular clinical characteristics, they show an association with the expression of lymphoid‐related markers. © 1995 Wiley‐Liss, Inc.
Variability in absolute lymphocyte counts obtained by automated cell counters
Authors: Simson E. , Groner W.
Published: June 2005

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)‐infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets.

We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built‐in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples.

Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory. © 1995 Wiley‐Liss, Inc.