Flow Cytometric Analysis Of Fluorescence Research Articles

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat.

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence insitu hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC-labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat-Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.

Abstract P5-05-07: Promestriene effects on estrogen-sensitive breast cancer cell proliferation in vitro

Abstract Introduction: Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with potential as a topical treatment for vaginal atrophy caused by estrogen deprivation. Topical Promestriene is minimally absorbed with chronic use and has been reported to significantly improve the symptoms of vaginal atrophy. However, to be considered for use for these symptoms in estrogen responsive post-menopausal breast cancer survivors on an aromatase inhibitor, Promestriene would need to show none of the cyto-proliferative properties of estrogen. Methods: We conducted in vitro testing of estrogen responsive breast cancer cell line MCF-7, T-47D, and BT-474 for dose-dependent, estrogen-like functional responses to Promestriene in both estrogen-rich and estrogen-deprived conditions. As assay controls, cultures treated with Fulvestrant, Testosterone, Progesterone and Estradiol were analyzed in parallel. Analysis: Dose dependent Estrogen-like responses in the cell lines were measured as 1) proliferation using flow cytometric analysis of CSFE fluorescence, 2) estrogen receptor expression by flow cytometry, and 3) GREB 1 RNA expression using TaqMan real time PCR quantitative analysis. All cultures were grown to quiescence in RPMI medium supplemented with 10% fetal calf serum. To induce estrogen deprivation, half of the cultures were then held 3 days in media pre-treated and containing anti-estrogen specific antibodies to sequester any residual estrogen from the culture. Data for each culture treatment were analyzed using linear regression analysis and compared using multifactorial or pairwise analyses to determine the significance of estrogen-like responses relative to the estradiol and untreated controls. Results: In estrogen sufficient cultures, Promestriene exhibited no cell proliferative properties on MCF-7, BT-474, or T47-D tumor cells, even at very high concentrations. However, when estrogen in the media was sequestered and culture allowed to quiesce without estrogen receptor stimulation, low dose Promestriene effects on cell proliferation in MCF-7 estrogen responsive cells were not significantly different from low dose estradiol. Low dose Promestriene (2-10pg/ml), unlike comparable doses of estradiol, did not stimulate GREB1 expression in MCF-7 cells. In contrast, T-47D and BT-474 cell lines proliferated and increased GREB- expression in response Promestriene or Fulvestrant treatments. Conclusions: The potential estrogen-like properties of Promestriene to stimulate the growth of estrogen receptor responsive breast cancer cell lines, especially in estrogen-deprived conditions, suggest caution when prescribed for vaginal atrophy in post-menopausal breast cancer survivors on an aromatase inhibitor. Its ability to activate growth and gene expression in ER- breast cancer cells warrants further study. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-07.

AGEs induce caspase-mediated apoptosis of rat BMSCs via TNFα production and oxidative stress

Diabetic humans and animals exhibit lower bone mass and healing, resulting from diminished bone formation. We have recently reported that type 1 diabetic rats have fewer bone marrow osteoprogenitor cells, and since the formation of advanced glycation end products (AGEs) in bone increases in diabetes, we explored possible mechanisms involved in AGE-induced apoptosis of rat bone marrow stromal cells (BMSCs). BMSCs isolated from 4-month-old rats were exposed to 10-400 μg/ml AGE-BSA for 16 h and apoptosis was quantified with PI/annexin V staining and flow cytometry. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. The formation of reactive oxygen species (ROS) was quantified by flow cytometric analysis of DCFDA fluorescence and the expression of genes by RT-PCR analysis. AGE-BSA at a concentration of 400 μg/ml increased the apoptosis of BMSCs two- to threefold, an effect completely blocked by a pan-caspase inhibitor. BSA or high concentrations of glucose had no effect. AGE-BSA-induced BMSC apoptosis was attenuated by a p38 inhibitor but not by an NF-κB inhibitor. Treatment with AGE-BSA induced the expression of several pro-apoptotic ligands and receptors, most notably tumor necrosis factor α (TNFα), TRAIL, lymphotoxin alpha, CD40, and TNFR2. Furthermore, AGE-BSA-induced apoptosis was completely blocked by pirfenidone, an inhibitor of TNFα production/secretion. Finally, AGE-BSA increased the production of ROS in BMSCs, and its apoptogenic effect was blocked by the antioxidant N-acetylcysteine (N-acetyl-L-cysteine). Thus, AGE-BSA increases the apoptosis of rat BMSCs via the activation of caspases, involving TNFα production/secretion, p38 MAPK signaling, and oxidative stress. We propose that increased protein glycation, such as that occurring under hyperglycemia, causes the apoptosis of BMSCs, which might significantly contribute to the development of osteopenia in diabetic animals.

221 FUNCTIONAL ANALYSIS OF A RARE MUTATION OF MITOCHONDRIAL DNA FOUND IN A PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010221 FUNCTIONAL ANALYSIS OF A RARE MUTATION OF MITOCHONDRIAL DNA FOUND IN A PROSTATE CANCER Rebecca Arnold, Adeboye Osunkoya, Sean O'Hearn, Fray Marshall, Douglas Wallace, Hong Yi, Elizabeth Wright, Carrie Sun, Les Costello, and John Petros Rebecca ArnoldRebecca Arnold Atlanta, GA More articles by this author , Adeboye OsunkoyaAdeboye Osunkoya Atlanta, GA More articles by this author , Sean O'HearnSean O'Hearn Irvine, CA More articles by this author , Fray MarshallFray Marshall Atlanta, GA More articles by this author , Douglas WallaceDouglas Wallace Irvine, CA More articles by this author , Hong YiHong Yi Atlanta, GA More articles by this author , Elizabeth WrightElizabeth Wright Atlanta, GA More articles by this author , Carrie SunCarrie Sun Atlanta, GA More articles by this author , Les CostelloLes Costello Baltimore, MD More articles by this author , and John PetrosJohn Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.279AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mitochondrial DNA mutations are present in prostate cancer patients both as modulators of inherited predisposition to disease and as somatically acquired mutations in the cancer. In addition, mtDNA mutations can markedly influence metastatic capability of tumor cells in animal models. One gene in particular (cytochrome oxidase 1 [COI]) has been linked to prostate cancer (PNAS 102:719-724, 2005). We describe a rare heteroplasmic mutation of this gene in a patient with prostate cancer and the functional consequences of the mutation. METHODS We performed complete gene sequencing of the COI gene in laser capture microdissected patient prostate cancer tissue, peripheral blood and lymphoblast cell lines derived from the patient. Comparison to reference sequence and large online databases was done to ascertain the likelihood of pathogenicity. The activity of the enzyme (cytochrome oxidase) encoded by this gene was determined by dual wavelength spectrophotometry and reactive oxygen generation was determined using flow cytometric analysis of DCF-DA fluorescence. Electron microscopy of mutant and wild type prostate cancer cells was done. RESULTS The highly conserved DNA base (T) at nucleotide position 6124 of the mitochondrial genome was found to be mutated (to C) resulting in the amino acid substitution Met74Thr. This alteration resulted in a 29% decrease in activity of cytochrome oxidase (for which the COI gene constitutes the catalytic core) and a 1.75-fold increase in reactive oxygen (superoxide anion) production. The mutation was found to be heteroplasmic (existing in both mutant and wild type forms in approximately equal amounts) in the patient cancer tissue. Electron microscopy revealed substantial morphologic derangement of the mitochondria only in the presence of mutation. CONCLUSIONS A rare mutation of the mitochondrial gene previously linked to prostate cancer has been found in a patient and studied functionally and structurally in the laboratory. The mutation causes severe morphologic alteration of mitochondrial ultrastructure. The presumed link between mtDNA mutation and carcinogenesis (increased production of reactive oxygen species) has been definitively confirmed for this discrete amino acid altering mutation. The CO1 mutation induces a dysfuntional cytochrome oxidase activity resulting in an increase in ROS. These results are demonstrative of the important role of altered mitochondrial oxidative metabolism in prostate and other cancers. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byAtala A (2018) Re: Bone Metastasis in Prostate Cancer: Recurring Mitochondrial DNA Mutation Reveals Selective Pressure Exerted by the Bone MicroenvironmentJournal of Urology, VOL. 196, NO. 3, (957-958), Online publication date: 1-Sep-2016. Volume 183Issue 4SApril 2010Page: e87 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rebecca Arnold Atlanta, GA More articles by this author Adeboye Osunkoya Atlanta, GA More articles by this author Sean O'Hearn Irvine, CA More articles by this author Fray Marshall Atlanta, GA More articles by this author Douglas Wallace Irvine, CA More articles by this author Hong Yi Atlanta, GA More articles by this author Elizabeth Wright Atlanta, GA More articles by this author Carrie Sun Atlanta, GA More articles by this author Les Costello Baltimore, MD More articles by this author John Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Rosiglitazone-induced SERCA2b inhibition: implications for monocyte cytoskeletal remodelling and diabetic microangiopathy

In type 2 diabetes, exposure of monocytic cells to inflammatory stimuli can lead to aberrant cytoskeletal remodelling and contribute to the development of diabetic microangiopathy. Previous studies within our research group have demonstrated that Rosiglitazone significantly reduces formyl-methionyl-leucyl-proline (f-MLP)-induced actin polymerization in monocytes by increasing cytosolic calcium ion concentrations ([Ca]i) in both resting and f-MLP-stimulated cells. As the timescale of this effect resembled inhibition of SERCA2b, the ‘housekeeping’ Ca2þ pump enzyme that sequesters Ca2þ into the ER compensatory to non-specific leakage into the cytoplasm, the present study sought to test the hypothesis that Rosiglitazone-induced increases in [Ca]i are brought about by inhibition of SERCA2b, and also investigated how such changes are transduced into cytoskeletal remodelling. Coupled-enzyme Ca2þ-ATPase assays revealed a SERCA2b activity of 49+ 5 nmol/mg/min in monocytic microsomes but pre-incubation with Rosiglitazone (20 mM; 30 min) induced statistically significant inhibition of this activity (P, 0.05). Permeabilization of cultured monocytes in external solutions containing differing [Ca]free was used to manipulate [Ca ]i, and F-actin content measured using flow cytometric analysis of FITC-phalloidin fluorescence. At [Ca]i .100 nM, a significant decrease in cells’ F-actin content was observed (P, 0.05). In conditions of minimal [Ca]i, f-MLP exerted a polymerizing effect on the actin cytoskeleton; however, as [Ca]i was increased to .100 nM, this polymerizing effect was significantly reduced. Finally, as a literature search identified the Ca2þ-dependent actin-modulating protein gelsolin as a candidate for transducing changes in [Ca]i into actin cytoskeletal remodelling, western blot experiments using anti-gelsolin antibodies were used to detect 88 kDa immunogenic bands in monocytic protein extracts, thus confirming the expression of gelsolin in monocytes. In conclusion, the current study demonstrates the importance of [Ca]i in modulating actin cytoskeletal remodelling, and suggests that Rosiglitazone, via its ability to affect monocytic Ca2þ signalling processes, may confer a level of protection against the development of diabetic microangiopathy.

Induction of necrosis and apoptosis to KB cancer cells by sanguinarine is associated with reactive oxygen species production and mitochondrial membrane depolarization

Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 microM) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (>52-58%) and growth of KB cancer cells at concentrations higher than 0.5-1 microM. Short-term exposure to sanguinarine (>0.5 microM) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 microM) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 microM) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death.

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow‐FISH‐DDD) to determine telomere length dynamics in proliferating cells

Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES1

Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured “free‐living” species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6–15 μm) possessed the lowest DNA content that we measured (1.5–4.8 pg·cell−1). Bloom‐forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.

Effects of Turkish pollen and propolis extracts on respiratory burst for K-562 cell lines

Bee-collected pollen and propolis are apicultural products which are composed of nutritionally valuable substances and contain considerable amounts of polyphenol substances which may act as potent antioxidants. We wanted to show if respiratory burst within a cancer cell lines could be influenced when incubated with pollen and propolis extracts or not. Pollen and propolis extracts at concentrations of 50, 25, 12.5 and 0 mg/ml were prepared by dimethyl sulfoxide (DMSO). K-562 cell cultures and mononuclear cell (MNC) cultures prepared from a peripheral blood sample to serve as control cells were incubated with extracts for 24 h. Determination of respiratory burst was carried out by intracellular dichlorofluorescein (DCFH) test by using flow-cytometric fluorescence analysis. While about 90% and 66% fluorescence was detected at zero concentrations for both K-562 and MNC cultures, fluorescence positivity decreased (between 3.8% and 11.8%) as concentrations of both propolis and pollen extracts increased for K-562 cell culture, but unchanged (between 20% and 83%) for MNC culture. It was concluded that pollen and propolis extracts inhibit respiratory burst within cancer cell lines probably by their antioxidant potentials.

193. Direct Comparison of Integration Efficiency and Stable Gene Expression Mediated by the Sleeping Beauty Transposon System and by the PhiC31 Phage Integrase System in Cultured Human Fibroblasts and in Various Stem Cell Targets

Top of pageAbstract Significant research efforts are directed toward achieving non-viral, DNA-mediated transfer and stable expression of genes in somatic cells for the treatment of diseases. Plasmid DNA has been successfully used to deliver genes into cells, but its usefulness is restricted by the inability to provide sustained expression. One alternative is to include components of an integrating system in the vector design. The Sleeping Beauty (SB) transposase and phiC31 integrase both bring about insertion of DNA sequences into the genomes of human cells, suggesting an alternative to viral transduction. Because each approach requires its own unique genetic elements and catalytic enzyme to mediate gene insertion into cellular chromosomes, we chose to directly compare the effectiveness of integration and stable expression using a two-component system that is internally controlled for the integrating sequence. The integrating vector (pKT2/NAG) consists of (i) a phiC31 integrase recognition site (attB), (ii) eF1-alpha regulated GFP expression unit, and (iii) neomycin resistance gene regulated by the PGK promoter, all flanked by (iv) T2 transposase binding sites (IR/DRs) separated by (v) a colE1 bacterial origin and kanR gene. Overexpression inhibition of the SB transposase suggests a potential limitation with respect to integration efficiency that can be achieved by this vector. To address this issue, we first studied the integrating efficiency of pKT2/NAG by evaluating the effect of the dose of the co-delivered enzyme-encoding plasmid on integration and stable gene expression as determined by G418-resistant colony formation and GFP fluorescence. In each case, pKT2/NAG (500 ng) was co-transfected with 150 ng, 500 ng, or 1500 ng of either CMV-regulated luciferase (pCMV-Luc), transposase (pCMV-SB), or integrase (pCMV-Int) expression plasmids into cultured human HT1080 cells. G418-resistant colony formation exceeded levels achieved by random integration (+ pCMV-Luc) at all tested concentrations of the SB transposase-encoding plasmid, with diminished efficiency at higher concentrations. The inverse relationship was observed for phiC31 integrase, where gene transfer improved as the concentration of pCMV-Int increased relative to pKT2/NAG. Drug-resistant clones have been isolated for studies into: (i) gene silencing by flow cytometric analysis of GFP fluorescence, (ii) insertional preference by cloning and sequencing vector::chromosome junctions, and (iii) insertion frequency by Southern analysis. We have also co-delivered both components into rat multipotent adult progenitor cells and found both systems to effectively mediate G418-resistant colony formation above background. Studies underway in mouse ES cells and in human CD34+ cells using this bifunctional approach will provide a broad assessment of the relative usefulness of these two major non-viral integrating systems for the purpose of ex vivo gene therapy applications in a range of human diseases.

Manipulation and Visualization of the Erythroid Lineage - In Vivo Models for Erythroid Disorders.

Red blood cells have a short half-life and are continuously renewed in a tightly controlled growth process. Dysregulation of erythropoiesis results in erythroleukemias or more frequently in anemias. To elucidate molecular mechanisms regulating red cell production in an animal model, we generated a mouse line (ErGFPcre) that simultaneously facilitates erythroid specific gene manipulation via the CreloxP recombination system and visualization of erythroid progenitor cells. To avoid site of integration effects frequently observed for randomly integrated transgenes, we used a knock-in strategy targeting the genomic EpoR locus that ensures a reliable EpoR promoter controlled transgene expression of our GFPcre fusion protein.The flow cytometric analysis of GFP fluorescence in different hematopoietic subpopulations of adult and embryonic ErGFPcre mice revealed a strictly erythroid-specific expression pattern for GFPcre. Further studies on GFP positive erythroid progenitor cells indicated a developmental switch in lineage progression from the hematopoietic stem cell compartment to early erythroid progenitor cells that are Sca-1 negative and c-kit high. To monitor previous and persistent GFPcre expression during development and to determine the efficiency of Cre-mediated recombination in our mouse model, we crossed the ErGFPcre mouse line with the LacZ reporter strain R26R. The spatial and temporal analysis of GFPcre-mediated LacZ expression confirmed that within the hematopoietic system GFPcre expression is limited to the erythroid lineage. Surprisingly, non-hematopoietic expression of GFPcre is restricted to the vascular system. It is possible that the observed differential transcriptional activity of the knock-in and the wild-type allele is linked to the absence of intron 2 to 7 in the knock-in locus. The quantitative analysis of the recombination frequency confirmed that GFPcre-mediated recombination is limited to erythroid progenitor cells and showed that it occurs in the adult bone marrow at a frequency of up to 80% and in the fetal liver with an efficiency close to 100%.Thus, our ErGFPcre mouse model offers the possibility to study regulatory mechanisms in erythroid progenitor cells and facilitates the establishment of mouse models for erythroid disorders.

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.

Adhesion of platelets to chemotactically responsive and non-responsive neutrophils

Aims-To investigate the heterotypic adhesion of unactivated platelets to chemotactically responsive (migrated) and non-responsive (non-migrated) polymorphonuclear neutrophils (PMN).Methods-Platelets and PMN were isolated from autologous, normal blood. Migrated and non-migrated PMN were separated after N-formylmethionyl-leucylphenylalanine (FMLP) stimulation. Platelets were labelled with a fluorescent monoclonal antibody directed against CD41 (GPIIb-IIIa). Platelets (3 x 10(8)/ml) and PMN (3 x 10(6)/ml) were incubated together. Heterotypic cell adhesion was measured in isolated PMN and PMN co-incubated with platelets by flow cytometric analysis of platelet marker fluorescence in PMN gated events. Platelet-PMN adhesion was also visualised by fluorescence microscopy.Results-In studies of isolated PMN, contaminating platelets were bound to 16-34% of unstimulated PMN, 7-22% of stimulated PMN, 2-4% of migrated PMN, and 17-24% of non-migrated PMN. When platelets were co-incubated with migrated or non-migrated PMN, 15-78% of PMN bound one or two platelets.Conclusions-Unactivated platelets adhere to isolated PMN in vitro. Fewer unactivated platelets were adhered to migrated PMN than to non-migrated PMN in isolated PMN preparations. These results indicate that platelets adhering to PMN are removed during PMN migration.

Evaluation of photosynthetic capacity in phytoplankton by flow cytometric analysis of DCMU-enhanced chlorophyll fluorescence

A new method is presented to evaluate the photosynthetic capacity of individual phytoplankton cells using flow cytometry. The method IS based on the well-known phenomenon in which chlorophyll a fluorescence is enhanced in the presence of 3-(3,4-dichloropheny1)-1,l-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. Using a mercury-cadmium arc lamp-based flow cytometer, w e found significant correlations between fluorescence enhancement and photosynthetic capacity. The correlation was dependent on species and growth irradiance. The regression coefficient ranged more than 5 times according to species, and cells under light-saturated growth were enhanced less than light-limited cells, although the former had higher photosynthetic capacity than the latter. The ability to measure DCMU-enhanced fluorescence in flow cytometry depends on a weak excitation beam and a long residence time in the sensing zone to ensure maximum variable fluorescence yield. We were unable to detect enhancement using laser-based flow cytometers. The variability in fluorescence enhancement is discussed in relation to its practical use in the field. The method was applied to natural assemblages in Bedford Basin (Canada): temporal variations of fluorescence enhancement were clearly evident in dominant diatoms and cryptophytes.

Flow cytometric analysis of the functional ability of neutrophils from patients with autoimmune neutropenia

Flow cytometry has been used to evaluate the functional ability of neutrophils and the expression of IgG Fc receptors (FcRII and FcRIII) in autoimmune neutropenia. Quantification of the neutrophil oxidative burst was made by assaying the production of 2'7'-dichlorofluorescein (DCF) from non-fluorescent 2'7'-dichlorofluorescein trapped within the cell, by flow cytometric analysis of cellular fluorescence. In the present study the DCF assay was used to examine the response of neutrophils to stimulation by opsonized and non-opsonized Staphylococcus aureus. In addition, the rate of uptake of S. aureus labelled with the red nuclear dye propidium iodide was determined. The presence of surface-bound immunoglobulin, which may affect the phagocytic capacity of the neutrophil, was also measured. No correlation between the neutrophil count and level of membrane-bound IgG or the rate of bacterial uptake was found. The studies were performed on twenty patients with autoimmune neutropenia, twelve with other autoimmune disorders and fourteen normal controls. The rate of uptake of bacteria was considered in relation to the expression of FcRII and FcRIII. Good correlation was found with the level of expression of FcRII, the major receptor for neutrophil activation, and the rate of uptake of bacteria (r = 0.64).

Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry

The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 μ M methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during g 1 and/or at G 1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G 2/M. To determine t-PA production CHO cells were sorted from G 1-, early/late S-, and G 2/M-phase. After 1-, 2-,and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production and secretion (2-h assay), unlike the expression of DHFR, increased continuously from relatively 100% in G 1 to 127% in early S and reached its maximum of 159% in late S, whereas in G 2/M-phase it decreased to 118%. Our results show that in pSVtPA.dhfr-coamplified CHO cells gene products DHFR and t-PA both exhibit different cell cycle-correlated accumulation and secretion, respectively, indicating that the brightest MTX-FITC-positive cells (G 2/M) do not display the highest t-PA secretion rate.

Flow cytometric analysis of doxorubicin accumulation in cells from human and rodent cell lines.

The correlation between intracellular accumulation of doxorubicin (DOX) and drug cytotoxicity was studied in cells from seven cell lines of rodent and human origin. We used three Chinese hamster ovary cell lines (AuxB1, CHRC5, and UV20), two murine tumor cell lines (KHT-LP1 and EMT6/Ro), and two human tumor cell lines (MGH-U1 and DLD-1). Intracellular DOX was measured by its fluorescence intensity with flow cytometry, and drug cytotoxicity was quantified with clonogenic assays. When data for all of the cell lines were combined, cell killing was correlated with the intracellular concentration of DOX (r = -.88). For the cell line AuxB1 and its DOX-resistant subline CHRC5, in which cells express high levels of P-glycoprotein, the relationship between cell survival and the intracellular concentration of DOX was stronger than that for the other cell lines. These results suggest that differences in intracellular accumulation of DOX account for much of the heterogeneity in response to the drug in cells from different cell lines, although additional mechanisms also contribute to variation in drug sensitivity. Flow cytometric analysis of intracellular DOX fluorescence is a simple assay that should be tested in cells from human tumors as a possible predictor of tumor response. For a given cell line, this technique also provides a rapid way to monitor the development of drug resistance after multiple courses of chemotherapy.

Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.

The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].

Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry.

Growth of human diploid fibroblasts in the presence of 5-bromodeoxyuridine, followed by flow cytometric analysis of DNA-specific fluorescence with Hoechst 33258 dye, allows quantitation of the proportion of cells that have not cycled, as well as those in G1 and G2 of two subsequent cell cycles. This technique allows rapid and accurate quantitation of the growth fraction and G1/S transition rate of these cells. The cell cycle kinetics of human diploid fibroblasts at all population doubling levels reveal two components: cycling cells showing a probabilistic rate of G1/S transition, and a variable proportion of noncycling cells. Both the transition probability (rate of exit from G1) and the noncycling proportion of cells change systematically as a function of serum concentration and as a function of population doubling level. The data suggest the existence of an underlying heterogeneity in the population of human diploid fibroblasts with respect to the capacity to divide in the presence of a given concentration of mitogen. Models of cell cycle kinetics must be modified to include regulation of growth by changes in the fraction of cycling cells, as well as by changes in the rate of exit from G1.

Flow cytometric analysis of cellular endogenous fluorescence simultaneously with emission from exogenous fluorochromes, light scatter and absorption

A cytofluorimeter capable of simultaneous 4-parameter analysis (i.e., endogenous fluorescence (NAD(P)H), exogenous fluorescent dyes, absorption and small angle scatter) is described. Analyses have been performed on isolated rat liver cells, white blood cells, bone marrow, thymus cells and yeast under different metabolic states. This allows correlations between cell function and concentration of cell substances, size, refractive indices, etc. Kinetic measurements are also possible.