Equipment
Charles University Faculty of Pharmacy and its Department of Pharmacology and Toxicology provide us with an appropriate technical background and core instruments including:
- cell line cryopreservation facility
- absorbance, fluorescence and luminescence Tecan Infinite M200 Pro readers
- centrifuges and ultracentrifuges
- Life Technologies QuantStudio 6 qRT-PCR apparatus
- Bio-Rad QX200 Digital Droplet PCR System
- Sony SA3800 spectral cell analyzer equipped with FACS
- Nikon TiE inverted fluorescence microscope equipped with Nikon A1 laser scanning confocal unit
- Perkin Elmer Liquid Scintillation Analyzer Tri-Carb 2900TR
- vivarium and laboratories for work with animals
Our laboratories, which are accredited for work with radioactivity and class II GMO, enable a wide range of experiments with both immortalized cell lines as well as primary cultures. They are equipped with several laminar flow cabinets, CO2 incubators, inverse microscopes and other essential cell biological tools.
Methods
In our studies, we employ number of cellular, molecular biological, biochemical, pharmacological, analytical and bioinformatic techniques. Specifically, we routinely use:
- accumulation studies with MDCKII cells overexpressing ABC or SLC transporters
- incubations with cytosolic and microsomal recombinant drug-metabolizing enzymes (cooperation with prof. Wsól)
- incubations with cells overexpressing drug-metabolizing enzymes (cooperation with prof. Guo)
- in silico molecular docking (cooperation with dr. Novotná)
- transient transfection
- MTT or XTT proliferation assays
- neutral red cytotoxicity assay
- caspase activity assays
- apoptosis detection using Annexin V/PI staining (flow cytometry) and staining of cell nuclei (fluorescence microscopy)
- drug combinations including quantification of combination effects using Chou-Talalay method
- bidirectional transport studies with MDCKII cells overexpressing ABC or SLC transporters
- quantification of drug concentrations in media and biological fluids using UHPLC coupled with QqQ mass spectrometer (cooperation with dr. Skarka)
- global proteomic analysis (cooperation with dr. Kupčík)
- gene expression and induction studies followed by qRT-PCR, dd-PCR or Western blotting analysis (cooperation with prof. Küpper)
- functional studies verifying results of induction studies
- siRNA-based knock-down assays
- development of drug-resistant cell lines using step-wise selection method
- migration- and extracelullar matrix-related experiments in 3D-structured tumoroids derived from immortalized cell lines (cooperation with prof. Lehti)
- experiments in 2D-structured primary explants derived from patient-donated non-small cell lung cancer biopsies (cooperation with dr. Hanke, M.D. and dr. Rozkoš, M.D.)
- experiments in 3D-structured tumoroids derived from patient-donated non-small cell lung cancer biospies and mice grafted with them (cooperation with prof. Tsao)
- design and preparation of samples for DNA-sequencing experiments
- bioinformatic signaling pathway cross-talk analysis (cooperation with prof. Lehti)
- bioinformatic analysis of cancer biology- and clinical treatment-related data from public databases
From the past, we have practical experience also with other methods such as pharmacokinetic studies in rats, molecular cloning, retroviral transduction, testing of transfection capabilities of nanostructured materials, etc.