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Biomedical subjects

I Rundquist

Publications and source records attributed to I Rundquist.

At least 19 recordsLinked to original sources
PubMed1995

High affinity binding of 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole to human neutrophilic granulocytes and lymphocytes.

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction method were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid-extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.

Binding Sites↗
PubMed1994

Lipofuscin-induced autofluorescence of living neonatal rat cardiomyocytes in culture.

Lipofuscin-specific autofluorescence of living, cultured rat neonatal myocardial myocytes was studied with respect to intensity and spectral characteristics. The autofluorescence emission spectrum changed over time indicating continuously ongoing intramolecular reorganisation of the pigment. Moreover, as compared with formaldehyde-fixed material native lipofuscin produced a considerable red-shifted autofluorescence, indicating formaldehyde-induced molecular modification or quenching of certain parts of the spectrum. The relationship between oxidative stress and lipofuscinogenesis was confirmed because the rate of lipofuscin accumulation was increased when cells were grown at increasing ambient oxygen concentrations.

Animals↗
PubMed1990

The use of acridine orange cytofluorometry in the study of macrophage lysosomal exocytosis.

We present a rather simple cytofluorometric technique for the study of exocytosis of lysosomal contents from individual cultured cells. It is based on the use of the lysosomotropic weak base acridine orange (AO) which, in its stacked form, as it occurs within lysosomes, emits red fluorescence when excited by blue light. Mouse peritoneal macrophages were cultured for 48 h and, after 2 h in serum-free medium, stained with AO. The cells were then exposed to F10-medium with or without newborn calf serum (NCS), zymosan A (Z) or cytochalasin B (CB) for different times at 20 or 37 degrees C. After staining, the macrophages showed no change in red fluorescence intensity, if stored at room temperature in the dark. If, however, the cells were kept in the incubator at 37 degrees C, the cells showed slightly decreasing red fluorescence intensity with time. This decrease was markedly potentiated by the presence of NCS, Z or CB, which are known to induce secretion of lysosomal enzymes from macrophages in vitro. Selective lysosomal enzyme release was confirmed biochemically during treatment with zymosan A. The technique presented here may be of value in further studies on the stimulation of, and the mechanisms behind, lysosomal exocytosis in cultured cells.

Acridine Orange↗
PubMed1989

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions.

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.

Acridine Orange↗
PubMed1989

Estimation of pH in individual alveolar macrophage phagolysosomes.

A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.

Animals↗
PubMed1988

Increased breakdown of phosphatidylinositol 4,5-bisphosphate is not an initiating factor for actin assembly in human neutrophils.

The present study examined the possible role of increased phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) breakdown in the regulation of actin assembly in human neutrophils. Tetracaine, a local anesthetic, was used since it has recently been proposed to inhibit the phosphorylation of phosphatidylinositol 4-phosphate to form PtdIns(4,5)P2. Surprisingly, it was found that incubation with tetracaine alone increased the breakdown of PtdIns(4,5)P2, measured as total inositol trisphosphate formation. This occurred without any rise above basal in the cellular content of filamentous actin. However, in the presence of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), tetracaine potentiated the chemotactic-induced increase of both inositol trisphosphate formation and actin polymerization. To further explore the relationship between increased PtdIns(4,5)P2 breakdown and actin polymerization, the activity of phospholipase C was depressed by lowering the cytosolic free calcium ion level or by incubating the cells with ionomycin. In these cells, fMet-Leu-Phe stimulation still raised the cellular content of filamentous actin to a level similar to levels in nontreated cells, despite the absence of PtdIns(4,5)P2 hydrolysis. Consequently, increased breakdown of PtdIns(4,5)P2 alone is not enough to initiate actin polymerization, nor is the polymerization of actin dependent on an increased PtdIns(4,5)P2 breakdown. However, we cannot exclude the possibility that increased turnover of phosphoinositides might act as a modulator of actin assembly.

Actins↗
PubMed1987

DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study.

Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease. In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974. Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch. Thirty-micron sections were cut from each plug for dewaxing and disintegration. The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry. In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern. No apparent correlation was found between the histopathological grade and the DNA ploidy. Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy. The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.

Acid Phosphatase↗
PubMed1987

Flow cytofluorometry of lysosomal acridine orange uptake by living cultured cells. Effect of trypsinization and starvation.

The uptake of the fluorescent, lysosomotropic weak base acridine orange (AO) by living cells in culture was studied by flow cytofluorometry. A mouse myeloma cell line (SP 2/0), growing in suspension, and an anchorage-dependent human malignant glioma cell line (U-251 MG), brought into suspension by trypsinization, were used. The consequences of trypsinization were also studied using static cytofluorometry. The lysosomal accumulation of AO by myeloma cells growing in suspension was found to be only moderately affected by starvation (i.e. incubation without medium change) for a period of up to five days. Trypsinization of the glioma cells after staining with AO caused pronounced release of the fluorescent dye while trypsinization before staining with AO did not significantly change the average lysosomal concentration of AO. We did, however, notice certain side effects of trypsinization in the form of both increased cellular green fluorescence and greater intercellular variability that reduce the validity of data obtained from cells detached by routine trypsinization. In conclusion, the condition of the lysosomal vacuome of living cultured cells growing in suspension may be studied by flow cytofluorometry after vital staining with the lysosomotropic weak base AO. Anchorage-dependent trypsinized cells, however, yield unsatisfactory results when examined in a flow cytofluorometer system and are better studied while still attached to their substratum, using static cytofluorometry.

Acridine Orange↗
PubMed1987

Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt.

We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.

Antibodies, Monoclonal↗
PubMed1987

Use of cytofluorometry to evaluate binding of antibodies to the cytoskeleton of cultured cells.

Immunocytochemistry is routinely used to examine the occurrence and distribution of cytoskeletal proteins in cells, but the results are usually evaluated visually and subjectively. Little use has been made of the potential the method offers for quantitative work. Here we report on application of cytofluorometry to quantify binding of antibodies to the cytoskeleton of U-251 MG human malignant glioma cells in culture. The results show that cytofluorometry is a simple and reliable procedure for: (a) determining the optimal concentrations of primary and secondary antibodies and other labeling reagents; (b) evaluating the binding specificity of commercial secondary antisera; and (c) evaluating the effect of different preparatory procedures on preservation of and binding of antibodies to cytoskeletal structures. Experiments with a monoclonal antibody to tubulin show that preservation of tubulin is very sensitive to the preparatory procedures used. Maximum labeling of tubulin in intact cells was obtained when the cells were pre-fixed with formaldehyde before permeabilization with solvent. Maximum labeling of tubulin in Triton-extracted cytoskeletons was achieved by pre-fixing the cells with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate), extracting with Triton in a microtubule-stabilizing buffer, and post-fixing with formaldehyde. GTP was not required to preserve tubulin in cytoskeletons.

Antibodies, Monoclonal↗
PubMed1985

Sciatic nerve blood flow measured by laser Doppler flowmetry and [14C]iodoantipyrine.

Blood flow was examined in sciatic nerves of pentobarbital-anesthetized rats by means of laser Doppler flowmetry (LDF) and intravenous [14C]iodoantipyrine infusion. Continuous LDF signals demonstrated slow oscillations and acute, pressure-related changes in flow. The steady-state LDF signal was related linearly to nerve blood flow, as measured with [14C]iodoantipyrine, in intact nerves and nerves stripped of the epineurium. In 14 intact nerves, nerve blood flow averaged 0.27 +/- 0.03 (SE) ml X min-1 X g-1, whereas it averaged 0.13 +/- 0.01 in 5 stripped nerves. Autoradiographs of [3H]-nicotine-infused nerves and intra-arterial injection of 57Co-labeled microspheres demonstrated that flow was not uniform throughout the nerve cross section. The results indicate that LDF can be used to examine nerve blood flow in vivo, demonstrate a linear relation between the LDF signal and flow, and establish absolute values for blood flow in intact and stripped nerves of the anesthetized rat.

Animals↗
PubMed1984

Dimethylsulfoxide increases the survival and lysosomal stability of mouse peritoneal macrophages exposed to low-LET ionizing radiation and/or ionic iron in culture.

Reactive oxygen-derived free radicals form excessively during irradiation of biological structures, but also normally in many cellular oxidative processes, albeit in small amounts. Unless scavenged by protective mechanisms, such radicals may induce peroxidation of polyunsaturated fatty acids resulting in membrane damage. The process may be catalysed to a considerable extent by transitional metals with the capacity to form redox systems, such as Fe3+ in equilibrium Fe2+. In the present study, it is shown that radiation by X-rays and/or exposure to ionic iron (Fe3+) causes decreased survival in parallel with lysosomal labilization of cultured mouse peritoneal macrophages (MPMs). The latter event was demonstrated as a reduced capacity of lysosomes in living MPMs to retain acridine orange during photo-oxidative stress caused by continuous exposure to blue light of short wavelength. The effects of X-irradiation, and/or lysosomal iron-loading, could be counteracted by the addition of the .OH-scavenging drug dimethylsulfoxide (DMSO) to the cell culture medium. The findings suggest that X-irradiation may damage certain sensitive G0 cells, such as Kupffer cells, serous cells of salivary glands and old macrophages, which normally have substantial concentrations of metals within their vacuolar apparatus, possibly by lysosomal damage involving .OH-mediated lipid peroxidation.

Acridine Orange↗
PubMed1984

Cytofluorometric quantitation of acridine orange uptake by cultured cells.

The vacuolar accumulation of the lysosomotropic weak base acridine orange (AO) within living cells in culture was studied by cytofluorometry. Mouse peritoneal macrophages, malignant human glioma cells, and normal human glial cells were utilized. Exposure to AO resulted in granular bright red fluorescence, as well as a diffuse weak green background fluorescence. To obtain reproducible "staining" conditions, the red granular fluorescence was measured as a function of dye concentration and staining time. Exposure to high concentrations of AO (greater than 10 micrograms/ml) was found to cause cell damage in combination with markedly changed fluorescence distribution for the cell population with reduced mean fluorescence and increased variability. Granular uptake of AO was pH-dependent and almost zero at pH 5.5. AO fluorescence, as measured by cytofluorometry, was found to be roughly linear to the amount of AO present in the cells, as measured by spectrofluorometry after cell solubilization, indicating negligible fluorescence quenching. AO labelling of living cells might serve as a useful indicator of the condition of the cellular vacuolar (lysosomal) apparatus.

Acridine Orange↗
PubMed1983

Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay.

Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. When extracellular pH was lowered a slight decrease in phagolysosomal pH was detected in macrophages but not in PMN. When measuring total fluorescence, interaction at pH 5.8 for PMN and at pH 4.4 for macrophages is recommended, since the intensity of extra- and intracellular fluorescence are equal under these conditions. Thirty different dyes were tested for dye exclusion and fluorescence quenching of FITC-conjugated yeast particles, and FITC-conjugated IgG. Because of the lysosomotropic effect of basic dyes, acid and direct dyes are preferable as quenching agents. We could not find physical or chemical properties of the dyes that correlated with their quenching effect. Heat aggregated IgG was used as an immune complex analogue in the development of the assay. Trypan blue (0.2 mg/ml) at pH 4.4 was found to be the best quenching agent of extracellular fluorescence when using ingested aggregated IgG. The technique offers a simple method of quantifying ingested protein aggregates and of studying heterogeneity in phagocyte populations.

Animals↗
PubMed1983

A simple fluorescence technique to stain the plasma membrane of human neutrophils.

Three different fluorochromes were tested for their ability to label the plasma membrane proteins of neutrophils without labelling intracellular structures. A fluorescence quenching technique was used to differentiate between extra- and intracellularly localized fluorescence. Fluorescamin and fluoresceinisothiocyanate were shown to stain intracellular structures as well as the plasma membranes of the cells. Another fluorochrome, Evans Blue, is proposed since this dye was shown, by using the fluorescence quenching technique, to selectively stain the plasma membrane of viable neutrophils.

Cell Membrane↗
PubMed1983

A versatile localization system for microscopic multiparametric analysis of cells.

A new, simple and relatively inexpensive electronic digital position readout (DPRO) system which can be applied to the rapid localization and recovery of microscopic material is described. It is based upon a commercially available digital position readout system which is routinely utilized by industry for small machine tools and measuring equipment. This has been mounted onto the stage of various microscopic instrumentation to provide X and Y coordinates relative to an arbitrary reference point. The integration of small computers interfaced to scanning interferometric, microdensitometric and fluorescence microscopes were used to demonstrate the reliability, versatility and ease of application of this system to problems of multiparametric measurements and analysis of cultured cells. The system may be expanded and applied to clinical material to obtain automatized, multiparametric measurements of cells in haematology and clinical cytology.

Cells, Cultured↗
PubMed1982

Uptake and turnover of dopamine in rat mast cells studied by cytofluorometry and high performance liquid chromatography.

Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injected L-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.

Animals↗