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PubMed1992

Differentiation of A31T6 proadipocytes to adipocytes: a flow cytometric analysis.

A flow cytometric assay has been developed which provides precise, quantitative information on the accumulation of cytoplasmic triglycerides in individual A31T6 proadipocytes as they differentiate into adipocytes. The opportunity to measure multiple optical parameters on a cell-by-cell basis has enabled us to monitor phenotypic aspects of differentiation with a greater level of sensitivity than was previously possible. Using the fluorescent hydrophobic probe, Nile red, we have found that as a cell proceeds along the differentiation pathway, the gold fluorescence signal from the cell increases, reflecting the accumulation of cytoplasmic lipid droplets. The value of the ratio of gold to red fluorescence defines a cell as being differentiated or undifferentiated. The greater resolution afforded by this cytometric method over more conventional approaches has allowed us to determine (1) the presence of an undifferentiated population of cells whose existence is not detected by conventional phase microscopy, (2) that insulin is not required to drive differentiation in this system, though the rate of differentiation is increased when the cells are exposed to insulin in combination with dexamethasone, (3) that exposure to a combination of insulin and dexamethasone results in a lower accumulation of lipid in a cell than does exposure to either agent alone, and (4) that A31T6 cells show the same response to differentiation-promoting agents whether applied at the time of plating or at confluence.

Adipose Tissue↗

PubMed1977

Quantitation of B cells in peripheral blood by polyacrylamide beads coated with anti-human chain antibody.

A method for quantitating peripheral blood B cells is described which utilizes anti-heavy chain specific antibody attached to polyacrylamide beads. The method has the advantages of ease of performance, of utilizing reagents which are commercially available and routine phase microscopy. In addition, the reagents are stable for prolonged periods of time and only small amounts are required. Utilizing this assay, the following percentages of B cells have been found in normals: IgG 6.3 +/- 0.6; IgM 4.3 +/- 0.5; IgA 4.0 +/- 0.5. The polyacrylamide beads may be phatocytized allowed recognition of certain phagocytic mononuclear cells.

Acrylamides↗

PubMed1982

A rapid method for isolation of human mononuclear cells free of significant platelet contamination.

In order to obtain mononuclear cells from peripheral human blood for the study of cell surface receptors, it was necessary to effectively eliminate contaminating platelets. The usual Hypaque-Ficoll isolation procedures were found to produce mononuclear cells contaminated with 10-1000 platelets per mononuclear cell (by phase microscopy). Multiple slow speed centrifugations reduced the contamination to 5-10 platelets per mononuclear cell. However, centrifugation of EDTA-anticoagulated blood through Hypaque (D20(20) 1.060) at 400 X g for 5 min at 22 degrees C followed by the usual Hypaque-Ficoll gradient reduced platelet contamination to less than 1 platelet per 2 mononuclear cells. Thus, a rapid and simple gradient procedure is capable of significantly reducing platelet contamination of mononuclear cell preparations and should facilitate the analysis of mononuclear cell receptors and functions.

Blood Platelets↗

PubMed1984

Effects of ethanol on cultured glial cells: proliferation and glutamine synthetase activity.

The development of glial-enriched cultures prepared from 15-day-old embryonic chick cerebral hemispheres was examined after exposure to ethanol. Between culture days 6 and 10, ethanol was added to the medium at 4 concentrations: 0.1%, 0.5%, 1.0% and 2.0% (w/v). During the drug exposure interval, morphologic maturation of the cultures was surveyed with phase microscopy; cell proliferation was assessed by cell counts and DNA content. Exposure to ethanol concentrations of 0.1% and 0.5% resulted in only minimal changes in the parameters investigated, whereas biosynthetic and enzymatic activities were altered at a threshold dose of 1.0% ethanol. A small number of cells resembling reactive astrocytes were encountered in cultures exposed to 1.0% and 2.0% ethanol. In these cultures the number of cells and DNA content were lower than in control cultures. Additionally, [14C]leucine incorporation into protein and glutamine synthetase activity were markedly diminished in cultures treated with 1.0% and 2.0% ethanol. The ethanol-related reduction in glutamine synthetase activity was not accompanied by an equivalent decrease in protein content, suggesting that glutamine synthetase or cells rich in this enzyme are selectively vulnerable to ethanol. Impairment in glutamate/glutamine metabolism may be a potential consequence of ethanol-induced changes in glutamine synthetase activity.

Animals↗

PubMed2004

An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction.

Alizarin red S (ARS) staining has been used for decades to evaluate calcium-rich deposits by cells in culture. It is particularly versatile in that the dye can be extracted from the stained monolayer and assayed. This study describes a sensitive method for the recovery and semiquantification of ARS in a stained monolayer by acetic acid extraction and neutralization with ammonium hydroxide followed by colorimetric detection at 405 nm. This method was three times more sensitive than an older method involving cetylpyridinium chloride (CPC) extraction and resulted in a better signal to noise ratio, especially for weakly stained monolayers. The assay facilitates detailed inspection of mineralization by phase microscopy and semiquantification of the entire monolayer by extraction and quantification. The sensitivity of the assay is improved by the extraction of the calcified mineral at low pH and, since the mineral is already stained in a quantitative manner, there is no requirement for an additional colorimetric quantification step. Furthermore, the linear range is much wider than those of conventional assays for calcium, making dilutions of mineral extracts prior to measurement unnecessary. It has a wide range of potential uses including tumor characterization, mesenchymal stem cell evaluation, and osteogenic compound screening. Although more labor intensive than CPC extraction, the protocol is more sensitive and yields more reliable results for weakly mineralizing samples.

Anthraquinones↗

PubMed2005

Constant decline in sperm concentration in infertile males in an urban population: experience over 18 years.

OBJECTIVE: To investigate semen quality in infertile men from an urban population over an 18-year period. DESIGN: Retrospective study. SETTING: Andrology clinic at a city university hospital. PATIENTS: A total of 9,327 men were referred to the clinic for infertility investigation. After excluding those with azoospermia, 7,780 samples were evaluated. INTERVENTION(S): Semen samples were analyzed within half an hour of production using computer-automated semen analysis and phase microscopy. MAIN OUTCOME MEASURE(S): Sperm concentration, morphology, and motility; semen pH and leukocyte concentration; and patient age. RESULT(S): The median patient age over the study period was 31.6 years, with an increase from 30.8 years in 1986 to 34.4 years in 2003. The median sperm concentration was 10.25 million/mL for the entire period, with a decline from 27.75 million/mL in 1986 to 4.60 million/mL in 2003. The median proportion of normally shaped spermatozoa was 15%, and the proportion of motile spermatozoa was 21%. The pH increased from 7.4 in 1986 to 7.9 in 2003, and the median leukocyte concentration was 1.50 million/mL. CONCLUSION(S): A constant decline in median sperm concentration was found in the infertile men. The reason for this remains unclear, although the high pH and leukocyte concentration indicates the involvement of infection.

Adult↗

PubMed1999

Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.

A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. The homophilic cell adhesion molecule contact site A, a lipid-anchored glycoprotein from cells of the slime mold Dictyostelium discoideum, is used as receptor. The success of the reconstitution, the structure and the dynamics of the model membranes are studied by various techniques including film balance techniques, micro fluorescence, fluorescence recovery after photobleaching, electron microscopy, and phase contrast microscopy. The interaction of the functionalized giant vesicles with the supported bilayer is studied by reflection interference contrast microscopy, and the adhesion strength is evaluated quantitatively by a recently developed technique. At low receptor concentrations adhesion-induced receptor segregation in the membranes leads to decomposition of the contact zone between membranes into domains of strong (receptor-mediated) adhesion and regions of weak adhesion while continuous zones of strong adhesion form at high receptor densities. The adhesion strengths (measured in terms of the spreading pressure S) of the various states of adhesion are obtained locally by analysis of the vesicle contour near the contact line in terms of elastic boundary conditions of adhesion: the balance of tensions and moments. The spreading pressure of the weak adhesion zones is S approximately 10(-9) J/m(2) and is determined by the interplay of gravitation and undulation forces whereas the spreading pressure of the tight adhesion domains is of the order S approximately 10(-6) J/m(2).

Adsorption↗

PubMed1984

Pseudothrombocytopenia: a laboratory artifact with potentially serious consequences.

When a low platelet count is unexpected or is unaccompanied by signs or symptoms of hemorrhage, pseudothrombocytopenia should be suspected. The platelet number and morphologic features should be examined on a smear prepared from blood anticoagulated with EDTA. Platelet counts should be repeated either by obtaining a finger-stock specimen with an ammonium oxalate Unopette and counting by phase microscopy or by collecting both an EDTA- and a sodium citrate-anticoagulated venous sample, performing a platelet count on both test tubes, and examining a blood smear from each test tube. If a Coulter Model S Plus IV or V or the Technicon H6000 is used for performance of platelet counts, examination of the histogram display of the Coulter instrument or the peroxidase X-Y display of the Technicon H6000 should alert the instrument operator to the presence of EDTA-induced platelet clumping and prevent the reporting of a spuriously low platelet count.

Clinical Laboratory Techniques↗

PubMed2000

Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its correlation with motility.

Methylene blue reduction rates (MeBRR) were evaluated spectrophotometrically to study bovine spermatozoal mitochondrial function and its relation to motility. A chemical reaction (H2SO4, methylene blue solution, and zinc powder) was used to quantify methylene blue reduction. Absorbance measurements were made for 10 min at 609 nm in a narrow band spectrophotometer. In a second experiment, fresh ejaculates were assessed for concentration and motility evaluated with phase microscopy (37 degrees C). Semen was diluted to 100 million cells/mL in a sodium citrate-glucose buffer and methylene blue. Absorbance and motility were evaluated every 30 min for 2.5 h in a water-jacketed cuvette (41 degrees C). Methylene blue reduction rates and motility decreased at each subsequent period. Methylene blue reduction rates were correlated to sperm motility. Lastly, the methylene blue reduction rate was measured with a broad band spectrophotometer and compared with motility using similar conditions. Motility estimates were made on sperm from the cuvettes. Sperm motility was correlated to methylene blue reduction rates measured spectrophotometrically.

Animals↗

PubMed1997

Control of tuberculosis by community health workers in Bangladesh.

BACKGROUND: Tuberculosis remains a major public-health problem in Bangladesh, despite national efforts to improve case identification and treatment compliance. In 1984, BRAC (formerly the Bangladesh Rural Advancement Committee), a national, non-governmental organisation, began an experimental tuberculosis-control programme in one thana (subdistrict). Community health workers screened villagers for chronic cough and collected sputum samples for acid-fast bacillus (AFB) microscopy (phase one). Positive patients received 12 months of directly observed therapy. Phase two (1992-94) included another nine thanas and, in phase three (1995), eight more thanas were included. From 1995, the treatment was an 8-month oral regimen. METHODS: In 1995-96, we analysed all programme data from 1992 to 1995. First we analysed phases two (12-month therapy) and three (8-month therapy) separately for proportion cured, died, treatment, failed, defaulted, migrated, and referred. Second, we did a cross-sectional survey of tuberculosis cases in more than 9000 randomly selected households in two phase-two thanas and one non-programme thana, and analysed the follow-up of all patients treated in the programme thanas. FINDINGS: In the phase-two analysis, 3497 (90%) of 3886 cases identified had accepted 12-month treatment. In phase three, all of 1741 identified cases accepted the 8-month regimen. 2833 (81.0%) and 1496 (85.9%) in phases two and three, respectively, were cured; 336 (9.6%) and 133 (7.6%) died. The relapse rate 2 or more years after treatment was discontinued was higher than the early relapse rate. The drop-out rate was 3.1%. In the cross-sectional survey, the prevalence of tuberculosis in the two programme thanas was half of that in the comparison thana, where only government services were available (0.07 vs 0.15 per 100 [corrected]). INTERPRETATION: The BRAC tuberculosis-control programme has successfully achieved high rates of case detection and treatment compliance, with a cure rate of at least 85% and a drop-out rate of 3.1%. The prevalence survey suggested that at least half of all existing cases had been detected by the programme.

Adolescent↗

PubMed1998

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage.

Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.

Animals↗

PubMed2005

Anticarcinogenic effects of glycoalkaloids from potatoes against human cervical, liver, lymphoma, and stomach cancer cells.

Methods were devised for the isolation of large amounts of pure alpha-chaconine and alpha-solanine from Dejima potatoes and for the extraction and analysis of total glycoalkaloids from five fresh potato varieties (Dejima, Jowon, Sumi, Toya, and Vora Valley). These compounds were then evaluated in experiments using a tetrazolium microculture (MTT) assay to assess the anticarcinogenic effects of (a) the isolated pure glycoalkaloids separately, (b) artificial mixtures of the two glycoalkaloids, and (c) the total glycoalkaloids isolated from each of the five potato varieties. All samples tested reduced the numbers of the following human cell lines: cervical (HeLa), liver (HepG2), lymphoma (U937), stomach (AGS and KATO III) cancer cells and normal liver (Chang) cells. The results show that (a) the effects of the glycoalkaloids were concentration dependent in the range of 0.1-10 mug/mL (0.117-11.7 nmol/mL); (b) alpha-chaconine was more active than was alpha-solanine; (c) some mixtures exhibited synergistic effects, whereas other produced additive ones; (d) the different cancer cells varied in their susceptibilities to destruction; and (e) the destruction of normal liver cells was generally lower than that of cancer liver cells. The decreases in cell populations were also observed visually by reversed-phase microscopy. The results complement related observations on the anticarcinogenic potential of food ingredients.

Anticarcinogenic Agents↗

PubMed2007

Structure-activity relationships of tea compounds against human cancer cells.

The content of the biologically active amino acid theanine in 15 commercial black, green, specialty, and herbal tea leaves was determined as the 2,4-dinitrophenyltheanine derivative (DNP-theanine) by a validated HPLC method. To define relative anticarcinogenic potencies of tea compounds and teas, nine green tea catechins, three black tea theaflavins, and theanine as well as aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). The growth of normal human lung (HEL299) cells was not inhibited. The destruction of cancer cells was also observed visually by reverse phase microscopy. Statistical analysis of the data showed that (a) the anticarcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent over the ranges from 50 to 400 microg/mL of tea compound and from 50 to 400 microg/g of tea solids; (b) the different cancer cells varied in their susceptibilities to destruction; (c) 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and (d) flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. The findings extend related observations on the anticarcinogenic potential of tea ingredients and suggest that consumers may benefit more by drinking both green and black teas.

Anticarcinogenic Agents↗

PubMed2003

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication.

This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium. Large and spacious PV with multiple non-replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.

Animals↗

PubMedDate not supplied

Circulation and function of human platelets isolated from units of CPDA-1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO.

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2-3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 +/- 17 percent (mean +/- one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 +/- 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 +/- 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin-prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.

Adenine↗

PubMed1994

A multicenter evaluation of reproducibility of swirling in platelet concentrates. Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion.

BACKGROUND: Eleven laboratories participated in a study investigating whether swirling can be used in monitoring platelet discoid morphology in platelet concentrates (PCs). STUDY DESIGN AND METHODS: In each laboratory, two readers observed the swirling pattern in 1- to 5-day-old PCs, in PCs containing spherical platelets obtained by 4 degrees C storage (spherical PCs), and in PCs containing known proportions of spherical and discoid platelets. Swirling was reported as positive, intermediate, or negative. Moreover, platelet morphology was evaluated by oil-phase microscopy. RESULTS: Swirling of spherical PCs was found to be positive and negative in 5 and 68 percent of cases, respectively. Conversely, swirling of fresh PCs was positive and negative in 83 and 2 percent of cases, respectively. Results in PCs containing known amounts of added spherical platelets were intermediate between those in fresh and spherical PCs. The observed agreement between the two readers (number of concordant readings/total readings) was 67 percent. Most disagreements concerned positive versus intermediate or intermediate versus negative evaluations, whereas a positive versus negative disagreement was observed in only 1 percent of cases. The percentages of discs found by microscopy showed greater variation among the different laboratories, indicating that this measurement should be better standardized. CONCLUSION: Evaluation of swirling seems promising for large-scale quality control of PCs. Further studies are needed to confirm this hypothesis.

Blood Platelets↗

PubMed1978

[On the aetiology of malignant pericardial mesothelioma (author's transl)].

Two cases of malignant pericardial mesothelioma are described. Morphologically they corresponded to the epithelial or fibrous type, respectively. In one patient, an old-metal dealer, an increased number of asbestos bodies were found in the lung parenchyma and phase-microscopy demonstrated increased asbestos needles in the lung parenchyma. Since, beyond a certain fibre size, asbestos can induce bronchial carcinoma as well as pleural or peritoneal mesothelioma, it is likely that the pericardial mesothelioma had a similar aetiology.

Aged↗

PubMed1991

[Donor tissue for keratoplasty. Report of experiences by the Hamburg cornea bank].

We report on or experience with the evaluation, preservation and clinical grafting of corneal donor tissue. From a total of 2274 documented donor corneae about 1535 specimens were subjected to a corneal preservation procedure. Refrigerated storage of donor tissue has been completely replaced by corneal tissue culture. On the basis of the tissue culture techniques of Sperling, improved methods to culture and evaluate donor tissue were developed. Using organ-culture as a preservation system, post mortem intervals for donor tissue could be extended to 50 hours. Organ culture in this respect serves as a viability test, as the donor endothelium is examined before culture and again before grafting. Thus, primary graft failures caused by degenerative endothelial cell changes could be avoided. Using phase microscopy, we developed criteria for the prognostic evaluation of corneal endothelium without vital staining. From a total number of about 1101 clinical grafts, 712 were taken from corneal preservation and 389 from refrigerated globes. Since 1985 exclusively tissue-cultured donor corneae are used for transplantation. A clinical follow-up of 327 patients showed cultured tissue to be at least equal to "fresh" corneal grafts.

Cell Survival↗