Micuda, Stanislav
Poslední změna: 19.07. 2019

Methods and equipment

The laboratories of LivPharm group at the Department of Pharmacology of Charles University, Faculty of Medicine are well equipped to enable in vivo and in vitrostudies of drug/endobiotic kinetics and dynamics together with detailed analysis of obtained samples by common analytical, biochemical and molecular biology methods.

Instruments

  • Quant Studio-7 qPCR (Thermo Fisher Scientific, San Jose, USA) for qRT-PCR nucleic acid analyses;
  • Simple WES automatic analyser and/or Bio-Rad electrophoretic and transblot units together with chemiluminiscent analyser Fusion Solo S  (Vilber, France) for Western blot protein analysis;
  • Agilent 1260 Infinity II HPLC system (Palo Alto, CA, USA);
  • IVIS Spectrum (Perkin Elmer) in vivo imaging system, Vetscan II biochemical analyser, inhalation anesthesia, syringe pumps, heated platforms and temperature controls forin vivo experiments;
  • Spark microplate reader with CO2module (Tecan, Grödig, Austria) for biochemical and in vitrocellular analyses;
  • Microscope BX51 (Olympus) with imageJ (National Institutes of Health, Bethesda, MD; http://rsweb.nih.gov/ij/) and NIS software analysis (Laboratory Imaging, Czech Republic) for histological evaluation.
  • Standard hematological and biochemical analyses of blood and serum samples from rats are performed by routine methods in Central laboratories of University Hospital using Sysmex XE-2100 analyzer (Sysmex, Kobe, Japan) and Modular PP analyzer (Roche, Basel, Switzerland), respectively.
  • LC-MS analyses are performed at cooperating Department of Biochemistry on a HPLC system Dionex Ultimate 3000 (Dionex Softron GmbH, Germany) and a triple quadrupole mass spectrometer TSQ Quantum Access Max with H-ESI II probe (Thermo Fisher Scientific, Inc., USA)

Methods

  • Animal studies.The kinetics of endogenous or exogenous substances is analyzed using rats or mice by sampling of blood, bile, urine, and stool and by harvesting the tissues at appropriate time points. Liver pharmacodynamics of current or potential therapeutic agents is studied on appropriate animal models of frequently ocuring disorders such as nonalcoholic fatty liver disease or cholestasis during pregnancy. All animals received humane care in accordance with the guidelines set by the institutional Animal Use and Care Committee of Charles University, Faculty of Medicine in Hradec Kralove, Czech Republic. The protocols of experiments are dicussed and approved by the same committee.  
  • To study liver impairment we analyse proper bioindicators in plasma, perform liver histological and immunohistological stainings, detect liver concentrations of reduced and oxidized glutathione, cholesterol and bile acids. BA concentrations in plasma, bile and stool are measured using the LC-MS method. 
  • Based on study, we analyze 30-50 genes (by qRT-PCR) and proteins (by Western blot) to uncover changes in enzymes, transporters, and their regulators implemented in lipidomics, and bile production. 
  • In vitro experiment are performed using HepaRG and HepG2 cells