Methods

In vitro studies are carried out using the following cell systems: cultured HUVEC, HL-60, U937 cell lines and whole blood or isolated neutrophils (dextran sedimentation, Ficoll separation, ammonium chloride lysis). In cultured cells, interactions of neutrophils/macrophages with epithelial cells and the effect of RONM donors/inhibitors or antiinflammatory drugs on the functions and interactions of leukocytes and epithelial cells are studied. Interactions of neutrophils, macrophages, epithelial cells in in vitro pathophysiological conditions (presence of pro-inflammatory cytokines, hypoxia/reoxygenation) are also studied.

In vivo studies are performed on the ischemia/reperfusion model of rat small intestine induced by the occlusion and subsequent release of the superior mesenteric artery.

Clinical studies are run under the collaboration with Center of Cardiovascular and Transplantation Surgery, Brno and University Hospital, Brno in patients undergoing open heart surgery, organ transplantation (heart, liver, kidney) or regular hemodialysis treatment.

Samples are analyzed using the following methods:

MethodParameter
Coulter-countercell numbers
Microscopycell numbers, cell viability, cell morphology
Spectrophotometrycell viability, metabolic activity as a measure of the number of viable cells, MPO activity, NO synthesis, individual antioxidants, lipid peroxidation, activity of specific enzymes
Flow cytometry

cell viability, cell cycle, oxidative burst of neutrophils, NO synthesis, surface molecule expression

Luminescencemetabolic activity as a measure of the number of viable cells, oxidative burst of neutrophils, MPO activity, NO synthesis, total radical-trapping antioxidant parameter, activity of specific enzymes

 


Chemiluminescence methods

Laboratory is equipped with two microtitre plate (Immunotech, Orion) luminometers.

  • Luminol- or lucigenin-enhanced chemiluminescence is used for the measurement of ROS production by different types of cells employing different activators (zymosan and opsonized zymosan particles, FMLP, PMA, calcium ionofore, starch grains).
  • Superoxide dismutase, catalase and horseradish peroxidase are used to differentiate between intra- and extracellularly generated oxidants. Sodium azide is used for the inhibition of extra- and intracellular MPO.
  • Continual 1-hr analysis of phagocytic chemiluminescent activity with consecutive dispensing of L-arginine and L-NAME is used to analyze the activity of nitric oxide synthase.
  • The application of antioxidants, NO donors (e.g.sodium nitropruside, SIN-1) and/or L-arginine analogues (L-NMMA, L-NAME, L-NNA) helps to differentiate between oxygen and nitrogen reactive metabolites.
  • Peroxyl radicals produced at a constant rate by thermal decomposition of ABAP are monitored by luminol-enhanced chemiluminescence. The total radical-trapping antioxidant parameter (TRAP) value is determined from the duration of the time period of the CL signal being diminished by serum antioxidants. When the serum antioxidants are exhausted, the CL signal is recovered. Trolox, a water soluble analogue of tocopherol is used as a reference inhibitor.
  • Luminometric assay for detection of MPO activity is based on bromide-dependent chemiluminescence in the presence of sample and hydrogen peroxide.
  • Luciferin-luciferase bioluminescence assay is used to determine cell proliferation and cytotoxicity in mammalian cells and to distinguish between cytostatic versus cytocidal potential of drugs on malignant cell growth. This assay is based on luciferase's requirement for ATP to produce light. In the presence of Mg+2, luciferase catalyzes the reaction of luciferin, ATP and oxygen to form oxyluciferin, AMP, carbon dioxide, pyrophosphate and light at 560 nm.

Flow cytometric methods

Flow cytometer FACS CALIBUR Becton Dickinson with sorting option.

  • Monoclonal antibodies labeled with different fluorescent probes are used for detection of surface antigen expression on leukocytes and endothelial cells.
  • Dichlorodihydrofluorescein diacetate is used to detect the generation of reactive oxygen intermediates in neutrophils and macrophages. Dichlorodihydrofluorescein diacetate reacts mainly with hydrogen peroxide and also reacts with peroxynitrite. Dihydrorhodamine 123 is the uncharged and nonfluorescent reduction product of the mitochondrion-selective dye rhodamine 123. This leuco dye passively diffuses across most cell membranes where it is oxidized to cationic rhodamine 123, which localizes in the mitochondria. Like H2DCF, dihydrorhodamine 123 does not directly detect superoxide, but rather reacts with hydrogen peroxide and also reacts with peroxynitrite. Dihydroethidium diffuses across cell membranes where it exhibits blue fluorescence; however, once this probe is oxidized to ethidium, it intercalates within the cell's DNA, staining its nucleus a bright fluorescent red as the probe is oxidized mainly by superoxide.
  • Nitric oxide is determined with the use of 1,2-diaminoanthroquinone, which is nonfluorescent until it reacts with NO to produce a red-fluorescent precipitate.
  • Cell cycle or cell viability is detected by flow cytometry after propidium iodide staining.

Spectrophotometric methods

Laboratory is equipped with microtitre plate (SLT.Rainbow) and micro-volume (NanoDrop) spectrophotometers.

  • Myeloperoxidase activity is determined using o-dianisidine.
  • The concentration of thiobarbituric acid reactive substance, and the index of lipid peroxidation is determined at 532 nm.
  • Uric acid is oxidized by oxygen in a reaction catalyzed by the enzyme uricase forming hydrogen peroxide and allantoin. Hydrogen peroxide is determined by oxidation coupling with 3,5-dichlor-2-hydroxybenzensulfonic acid (disodium salt) and 4-aminoantipyrine. This reaction is catalyzed by the enzyme peroxidase.
  • L-Ascorbic acid reduces the tetrazolium salt MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] in the presence of the electron carrier PMS (5-methylphenazinium methosulphate) at pH 3.5 to a formazan. Another method used in our laboratory is based on the reduction of ferric chloride (Fe3+) by ascorbic acid to ferrous ion (Fe2+) and a reaction with 2,4,6-tripiridil-S-triazina (TPTZ) developing a violet chromophore.
  • 5,5-dibromo-o-cresolsulphonphtalein (BromoCresol - BCP) forms a complex together with albumin in slightly acidic medium and in the presence of surfactans.
  • Bilirubin reacts with diazotized sulphanilic acid and forms an azo-dye product.
  • Nitric oxide determination by the Griess reagent: The Griess reagent provides a colorimetric assay for nitrites, and nitrates that have been reduced to nitrites, with a detection limit of about 100 nM. Nitrites react with the Griess reagent to form a purple azoderivative that can be monitored by absorbance at 548 nm.
  • MTT is used for detection of cellular redox potential for viability, cytotoxicity and proliferation assays. Following reduction, the water-soluble, colorless compound forms uncharged, brightly colored formazan.

Fluorescence methods

Laboratory is equipped with combined cuvette/microtitre plate (Tecan) spectrofluorometer/spectrophotometer.