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Results for “Laser Scanning Cytometry”

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PubMed2003

Nitrosative stress induced cytotoxicity in Giardia intestinalis.

AIMS: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. METHODS AND RESULTS: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. CONCLUSIONS: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.

Animals↗

PubMed1999

Germination of Bacillus anthracis spores within alveolar macrophages.

The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.

Animals↗

PubMedDate not supplied

Effects of novel safrole oxide derivatives, 1-propyl-3-(3,4-methylenedioxyphenyl)-2-propanol and 1-isopropoxy-3-(3,4-methylenedioxyphenyl)-2-propanol, on apoptosis induced by deprivation of survival factors in vascular endothelial cells.

Two safrole oxide derivatives, 1-propoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (FOD) and 1-isopropoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (GOD), were newly synthesized as promoters of apoptosis in vascular endothelial cells. The purpose of this study was to investigate the effects of these two safrole oxide derivatives on cell growth and apoptosis induced by deprivation of survival factors (serum and fibroblast growth factors, aFGF and bFGF) in vascular endothelial cells (VECs). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) method, agarose gel electrophoresis, laser scanning confocal microscopy, flow cytometry (FCM), and immunofluorescence assay were used. The cells deprived of FGF and serum were exposed to FOD or GOD 30 to 90 mg x L(-1) for 24 h, cell growth was suppressed (p < .05), whereas detachment and DNA fragmentation of these cells were promoted (p < .01). When the cells were treated with FOD 90 mg x L(-) for 24 h, apoptosis rate was 14.99% (p < .01). There were more cells in G2-M phase and less cells in S phase. At 90 mg x L(-1) concentration, GOD blocked 77.03%of the cells at G0-G1 phase., P53 level in VEC exposed to FOD or GOD was increased (p < .01). The data suggested that FOD and GOD might promote apoptosis of VEC by affect the cell cycle distribution, whereas P53 was involved in this pathway.

Apoptosis↗

PubMed2004

The plasma membrane of microaerophilic protists: oxidative and nitrosative stress.

The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC(4)(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC(4)(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O(2)-sensitive organisms and allows DiBAC(4)(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.

Animals↗

PubMed2005

Tyroserleutide tripeptide affects calcium homeostasis of human hepatocarcinoma BEL-7402 cells.

This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism preliminarily. YSL, at doses of 80 microg x kg(-1) x d(-1), 160 microg x kg(-1) x d(-1) and 320 microg x kg(-1) x d(-1) significantly inhibited the growth of the human hepatocarcinoma BEL-7402 tumor in nude mice, producing inhibition of 21.66%, 41.34%, and 34.78%, respectively. Ultra structure of BEL-7402 tumor in nude mice showed that YSL could induce tumor cells apoptosis and necrosis, cell organelle mitochondria and endoplasmic reticulum damage, and calcium overload. By confocal laser scanning microscopy and flow cytometry, we found that 10 microg/mL YSL rapidly induced an increase of the concentration of cytoplasmic free calcium in BEL-7402 cells in vitro, and maintained high concentrations of cytoplasmic free calcium for 1 h. Then the calcium concentration began to decrease after 2 h, and was lower than that of the control group at 4 h and 24 h (p < 0.05). YSL also decreased the mitochondrial transmembrane potential of BEL-7402 cells in vitro, but had no effect on the calcium homeostasis or mitochondrial transmembrane potential of Chang liver hepatocytes. So affecting calcium homeostasis, then inducing apoptosis and necrosis may be a mechanism by which YSL inhibits the tumor growth in animal model.

Animals↗

PubMed2005

ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells.

ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription-polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony-forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and DeltaNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%-3.0% positive cells by flow cytometry. The ABCG2-positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2-negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and DeltaNp63 mRNA and greater CFE than the non-SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2-positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2001

Morphometric characterization of endometrial glands using quantitative cytology.

OBJECTIVE: To determine the usefulness of the combination of confocal laser scanning microscopy (CLSM), image cytometry and three-dimensional (3D) imaging for analyzing architectural changes indicative of endometrial hyperplasia and grade 1 adenocarcinoma. STUDY DESIGN: Papanicolaou-stained endometrial samples (n = 180) were analyzed for specific cellular characteristics and analyzed by CLSM. Confocal images were obtained and then analyzed cytometrically and used for 3D reconstruction. RESULTS: Values obtained after image cytometry and 3D imaging increased significantly (P < .01) with the degree of cellular atypia. CONCLUSION: The combination of CLSM, image cytometry and 3D imaging is a valuable method for differential diagnosis of endometrial hyperplasia and grade 1 adenocarcinoma.

Adenocarcinoma↗

PubMed2005

[Effect of new photosensitizer CDHS801-mediated photodynamic therapy on bladder cancer: an experimental study].

OBJECTIVE: To investigate the effects of dynamic photodynamic therapy (PDT) on bladder cancer. METHODS: Human bladder cancer cells of the line T24 were co-cultured with CDHS801, a photosensitizer, and MitoTracker RED CMXRose and MitoTracker GREEN FM, mitochondria specific fluorescence probe dyes. Laser scanning confocal fluorescence microimaging system was applied to collect the fluorescence of the photosensitizer and the probes. T24 cells were cultured and divided into 4 groups: Group 1 as blank control group, Group 2 undergoing laser irradiation, Group 3, added with CDHS801 for 6h, and Group 4 (PDT group), added with CDHS801 and undergoing laser irradiation. The survival of the cells was examined by MTT colorimetric assay. The morphological changes and apoptosis of the photo-activated T24 cells were investigated by transmission electron microscopy, confocal laser scan microscopy, and flow cytometry. RESULTS: The fluorescence of the photosensitizer and that of the probes were detected in the cytoplasm and in the peri-nuclear region, mainly in the mitochondria, of the T24 cells. 2. The inhibitory rates of PDT on T24 cells were 0%, 7.3%, 10.8%, and 71.4% in the control group, Group 1, Group 2, and Group 3 respectively. T24 cell photo-activated with CDHS801 showed cell size shrinkage, condensed chromatin and formation of apoptotic body. Flow cytometry showed that apoptosis was seen in 55.31% of the photo-activated cells and peaked in the sub-G1 phase. However, no such changes were seen in the control group. CONCLUSION: CDHS801-based PDT can kill bladder cancer T24 cells. CDHS801 is localized in the cytoplasm and peri-nuclear region, mainly mitochondria, of tumor cell. CDHS801 based PDT maybe eliminate the T24 cell by the induction of apoptosis.

Apoptosis↗

PubMed2006

[Preparation and characterization of monoclonal antibodies against human secretory leukocyte protease inhibitor].

AIM: To prepare and identify monoclonal antibodies(mAbs) against human secretory leukocyte protease inhibitor (hSLPI). METHODS: BALB/c mice were immunized with hSLPI, and hybridoma cell lines were obtained by fusing mouse spleen cells with myeloma NS-1 cells. The specificity of mAbs were characterized by ELISA, Western blot, immunohistochemical staining, flow cytometry(FCM) and confocal laser scanning microscopy(CLSM). RESULTS: Four hybridoma cells which secreted the mAbs to hSLPI were obtained. 4 mAbs were IgM. Western blot analysis showed that the mAbs could recognize a target molecule with relative molecular mass of 12 000. Immunohistochemical staining revealed that the reactivities of 4 mAbs to the epithelial cells in lung and colon tissues, mast cell-like cells in lung, colon, tonsil and prepuce tissues were positive. The result of FCM showed that the 4 mAbs recognized SLPI expressed in A549 cells. CLSM examination confirmed that the fluorescent markers were mainly localized in the cytoplasm of A549 cells. CONCLUSION: The mAbs against hSLPI are prepared successfully, which provides valuable tool for studies on allergic and inflammatory diseases.

Animals↗

PubMed1996

Intracellular CD22 rapidly moves to the cell surface in a tyrosine kinase-dependent manner following antigen receptor stimulation.

CD22 is a key accessory molecule for Ag receptor signaling in B cells that becomes tyrosine phosphorylated in the signaling process. CD22 associates with sIg and strongly amplifies sIg-induced signals. During B cell development, CD22 is initially expressed intracellularly, with surface expression appearing with IgD expression. We used confocal laser-scanning microscopy and flow cytometry to analyze CD22 translocation responses to signaling events. Cross-linking surface IgM (sIgM) induced rapid movement of CD22 to the cell surface in both Ramos and Daudi B cells, with a 50 to 100% increase in surface expression observed within 5 min of stimulation. The increase in CD22 surface expression was specific in that CD19 expression was not affected. Both cell surface and intracellular CD22 were directed toward the site of sIgM stimulation. Treatment with the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV) also increased CD22 surface expression. The tyrosine kinase inhibitor tyrphostin A10 inhibited CD22 movement at concentrations that inhibited tyrosine phosphorylation of CD22 and other cellular proteins. In contrast to the B cell lines, mature peripheral blood B cells contained very little intracellular CD22 and showed no significant increase in surface expression following sIgM stimulation. The rapid directed movement of intracellular CD22 provides a new mechanism to dynamically regulate Ag receptor signaling, as well as CD22-mediated adhesion.

Antigens, CD↗

PubMed2002

Differences in the subcellular localization of alpha1-adrenoceptor subtypes can affect the subtype selectivity of drugs in a study with the fluorescent ligand BODIPY FL-prazosin.

G protein-coupled receptor (GPCR) subtypes are differentially distributed in the cell; however, it remains unclear how this affects the subtype selectivity of particular drugs. In the present study, we used flow cytometry analysis with the fluorescent ligand, BODIPY FL-prazosin, to study the relationship between the subcellular distribution of subtype receptors and the subtype-selective character of ligands using alpha1a and alpha1b-adrenoceptors (ARs). Alpha1a-ARs predominantly localize inside the cell, while alpha1b-ARs on the cell surface. Flow cytometry analysis and confocal laser-scanning micrographs of living cells showed that BODIPY FL-prazosin can label not only alpha1-ARs on the cell surface, but also those localized inside the cell. Furthermore, flow cytometry analysis of alpha1A-AR-selective drug, KMD-3213, and alpha1B-AR-selective drug, CEC, revealed that the major determinant of the subtype selectivity of each drug is different. The alpha1A-AR selectivity of KMD-3213 can be explained by its much higher affinity for alpha1a-AR than alpha1b-AR (affinity-dependent selectivity), while the alpha1B-AR selectivity of the hydrophilic alkylating agent CEC is due to preferential inactivation of alpha1-ARs on the cell surface (receptor localization-dependent selectivity). This study illustrates that factors in addition to the affinity of the drug for the receptor, such as subcellular localization of the receptor, should be taken into account in assessing the subtype selectivity of a drug.

Adrenergic alpha-Antagonists↗

PubMedDate not supplied

Structure property analysis of pentamethine indocyanine dyes: identification of a new dye for life science applications.

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.

Algorithms↗

PubMed2005

[Study on mechanism of tea polyphenols in inducing human lung cancer cell apoptosis in vitro].

OBJECTIVE: To investigate the apoptosis inducing effect of tea polyphenols (TPP) on human lung cancer cell (LCC) and its associative mechanism. METHODS: The apoptosis inducing effect of TPP on LCC in vitro, and its influence on expression of the related gene were determined by MTT assay, laser scanning confocal microscopy and flow cytometry. RESULTS: TPP in different concentration (50,100,200 and 400 microg/ml) had dose-dependent inhibitory effect on LCC, the inhibitory rate was 28.69+/-1.27% ,46. 19+/-1.79% ,64.61+/-1.29%, 75.90+/-1.96%, respectively. The inhibited LCC were blocked in (G0/G1 phase, and could not transferred to S and G2/ M phase of cell cycle. Meanwhile, TPP could induce apoptosis of LCC, the apoptotic rate being 4.76+/-0.11 %, 5.78+/-0.38 %, 10.06+/-0.67 %, 24.44+/-0.44 %, respectively. Morphologic changes of cells were seen in laser scanning confocal microscopy observation. Compared to the control group, intracellular Ca2+ concentration, Annexin V expression, phospatase and tensin homologe deleted on chromosome ten (PTEN) protein and expression gradually increased, while Cyclin D1 protein expression gradually decreased in the TPP treated groups along with the increasing of TPS concentration. CONCLUSION: TTP can induce LCC apoptosis, the mechanism is related to the change of intracellular Ca2+ concentration, PTEN protein and Cyclin D1 protein expression.

Annexin A5↗

PubMed2000

[Apoptosis of human gastric cancer cell line MGC-803 induced by glycyrrhiza uralensis extract].

OBJECTIVE: To study the apoptosis in human gastric cancer cell MGC-803 induced by the extract of glycyrrhiza uralensis Fisch. METHODS: Apoptosis was detected by laser scanning confocal microscope, agarose gel electrophoresis and flow cytometry. The p53 gene expression was analyzed by double immunolabelling and immunohistochemistry technique. RESULTS: Chromatin condensation of different phases of apoptotic cells were observed by laser scanning confocal microscope, the percentage of apoptosis determined by flow cytometry was time- and dose-dependent, agarose gel electrophoresis of DNA exhibited obvious "ladder" in the cells treated with higher concentration of the extract of glycyrrhiza uralensis. The p53 gene expression was unchanged after treatment. CONCLUSION: Glycyrrhiza can induce human gastric cancer cell line MGC-803 to apoptosis by p53-independent pathway and can be used as a potential, natural apoptosis-inducing agent for gastric cancer therapy.

Apoptosis↗

PubMed2002

Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions.

Classical enumeration methods for anaerobes are time-consuming and require special conditions. Solid phase cytometry (SPC) is a recent laser scanning technique for the quantitative detection of fluorescently labelled bacteria on a membrane filter that eliminates the need for a growth phase. Fluorescent labelling of cells results from the cleavage by intracellular esterases of a fluorescein type ester to yield a free fluorescein derivative, which is retained only in cells with an intact cytoplasmic membrane. However, as the standard labelling procedure is carried out under the conditions of aerobiosis, labelling of anaerobic bacteria does not appear to be obvious. We have labelled eight strains of vegetative anaerobic bacteria (i.e. Bacteroides thetaiotaomicron, Clostridium bifermentans, C. butyricum, C. perfringens, Fusobacterium nucleatum, Porphyromonas canoris, P. gingivalis, Propionibacterium acnes) and two strains of spores (C. butyricum, C. perfringens,) within 4 h under aerobic conditions. However, anaerobiosis remained necessary for spores of C. sordellii, C. sporogenes, C. tyrobutyricum. For vegetative cells of all strains, plots of SPC versus plate counts were linear with slopes exceeding 1.0, indicating that SPC consistently yielded higher numbers of bacteria.

Aerobiosis↗

PubMed2006

The nonopsonic allogeneic cell phagocytosis of macrophages detected by flow cytometry and two photon fluorescence microscope.

Phagocytosis, one of the apparent functions for macrophages, represents an early and crucial event in triggering host defenses against invading pathogens as well as allo- or xenogeneic rejection. Now, some methods have been used in detecting the opsonic phagocytosis of macrophages in xenogeneic settings. Efficient nonopsonic phagocytosis analysis method has not been established yet. In the present studies, allogeneic lymphocytes pre-labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) or derived from green fluorescent protein transgeneic B6 mice (GFP-B6 mice) were co-incubated with primary murine peritoneal macrophages (PEMs) for 1-2 h or were injected into murine peritoneal cavity for 30 to 240 min. Assays by flow cytometry (FCM) and two photon laser scanning microscope (TPM) showed an efficient uptake of both allogeneic lymphocytes and xenogeneic chicken red blood cells. The continuing process of nonopsonic phagocytosis of allogeneic lymphocytes by PEMs was recorded by TPM. Furthermore, the phenotype differences of PEMs with or without phagocytosis of allogeneic cells were determined by three-color FCMs. Significantly upregulated expressions of CD11b, CD44, TLR2 and TLR4 on PEMs were observed as early as 6 h after phagocytosis of allogeneic cells. Our present data indicated that the FCM and TPM combined method is a practical approach to detect macrophage nonopsonic phagocytosis of allogeneic lymphocytes and to identify the phenotype alteration of macrophages after phagocytosis.

Animals↗

PubMed1996

Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes.

We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials. Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate. Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections. The 3-D images were obtained by assembling these sections using a computerized image analysis device. The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere. The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase. The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase. The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.

Biomarkers↗

PubMed2004

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases.

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.

Biodegradation, Environmental↗