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MDR MODULATION TESTS
MDR modulation assays identify compounds that increase the sensitivity of cancer cells to treatment. These assays investigate different mechanisms of resistance such as efflux pumps, apoptotic pathways and cellular metabolism. Typical approaches include combination assays with chemotherapeutics, efflux pump inhibitors, apoptosis modulators and metabolism modulators. MDR modulation assays are a key tool for identifying new drug combinations and personalised medicine in the fight against tumour resistance.
Instrumentation: Automated pipetting station Biomek FX Laboratory Workstation (Beckman Coulter Life Sciences, USA)
ACCUMULATION TESTS
Accumulation tests are laboratory methods used to test the activity of efflux pumps in cells. Efflux pumps are transport proteins that actively remove substances, such as toxins or drugs, from cells. These tests use fluorescent compounds to assess the functionality of these pumps. In our laboratory, we study the activity of the efflux pumps P-glycoprotein and BCRP.
Instrumentation: automatic cell counter Cellometer Luna-II (Logos biosystems, South Korea)
WESTERN BLOT
Western blot is a laboratory technique used to detect specific proteins in a sample. Proteins are first separated by size by gel electrophoresis, then transferred to a membrane. Specific antibodies are applied to the membrane, which bind to the target protein and are then detected using an enzyme coupled to a secondary antibody. The resulting signal is visualised, allowing identification and quantification of the target protein.
ELISA
ELISA (enzyme-linked immunosorbent assay) is an analytical method for the quantitative determination of various antigens. It is based on the highly specific interaction of an antigen and an antibody with an enzyme (usually peroxidase or alkaline phosphatase) covalently bound to one of the partners. The enzyme catalyses the chemical conversion of a substrate added to the reaction mixture into a coloured product. This is then measured spectrophotometrically.
Instrumentation: HydroSpeed washer (Tecan, Switzerland), Infinite F Nano+ microplate reader (Tecan, Switzerland).
CYTOTOXICITY
Cell lines derived from human tissues are used to monitor toxicity, most commonly dermal fibroblasts or keratinocytes for substances intended for dermal application and renal cells for oral administration. Antitumour activity is then monitored as the ratio of cytotoxicity against the control cell line and against the tumour cell line. Through extensive national and international collaborations, the laboratory has a rich collection of tumour cell lines that are resistant to chemotherapeutic agents, making it possible to monitor collateral sensitivity, i.e. increased cytotoxicity of agents against a drug-resistant versus a drug-sensitive cell line. Cytotoxicity is usually monitored using the standard resazurin method, which is based on cell metabolic activity, where living cells metabolise resazurin to a fluorescent product.
Instrumentation: SpectraMax i3x MiniMax multimodal microplate reader (Molecular Devices, USA), Dispensor liquid and cell dispenser (BioTech, USA).
LUCIFERASE REPORTER ASSAYS
Luciferase reporter assays are laboratory techniques used to measure gene expression and study promoter activity. They use the enzyme luciferase, which catalyses a reaction that results in the emission of light when a substrate is added. The light signal is quantified by a luminometer, providing information about the activity of a particular promoter or gene expression. These assays are used to study promoter activity, signalling pathways and gene regulation. In our laboratory we focus on promoters associated with the transcription factors NFΚB, Nrf2 and p53.
Instrumentation: Temperature controlled incubators with CO2 atmosphere (Memmert, Germany; ThermoFisher Scientific, USA), Laminar Boxes (BioAir, Spain).
POLYMERASE CHAIN REACTION (PCR)
The Polymerase Chain Reaction (PCR) is a method used to amplify specific stretches of DNA. The process involves three steps: denaturation of the DNA, annealing of the primers and extension of the target region, which are repeated in several cycles. This method allows a large number of DNA copies to be made quickly and accurately, even from a small initial sample. It is used in the cloning, identification and production of proteins and other molecules. The second generation of this method - quantitative PCR with template DNA complementary to the original RNA (RT-qPCR) - allows the quantification of the transcription of various bacterial and tumour genes, especially efflux pump genes.
Instrumentation: CFX96 thermocycler (Bio-Rad, USA), OT-2 pipetting station (Opentrons, USA).
AGAROSE ELECTROPHORESIS
Agarose electrophoresis is a method used to separate biomolecules (DNA, RNA) according to their size and charge. Samples pipetted into wells in an agarose gel are driven by an electric field through the gel towards a positively charged electrode. Smaller molecules pass through the pores of the gel faster than larger molecules, resulting in size separation. Specific dyes are then used to visualise the samples in the gel (electrophoreogram), from which the size of the biomolecule, for example, can be determined. It is used for PCR control and biomolecule size determination.
MICRODILUTION METHOD
The microdilution method (ISO 20776-1) is a quantitative laboratory method for expressing the susceptibility of microorganisms to antimicrobial agents. A series of dilutions of the test substance are prepared in microtiter plates to which standardised amounts of microorganisms are added. After incubation, the growth of micro-organisms in each well is assessed. This method is suitable for determining the minimum inhibitory concentration (MIC) and for determining the efficacy of antibiotics and other antimicrobial agents.
Instrumentation: electronic pipettes (Eppendorf, Czech Republic)
TRANSFORMATION OF ESCHERICHIA COLI CELLS WITH A PLASMID CARRYING AN ANTIBIOTIC RESISTANCE GENE
A plasmid carrying a resistance gene (most commonly a gene for a destructase) is prepared in vitro by PCR and Gibson assembly. The plasmid is transformed into E. coli cells by heat shock. This culture is then used to screen for inhibitors of the resistance mechanism and potential adjuvants.
Instrumentation: Thermomixer (LAB MARK a. s., Czech Republic), T-Personal Thermocycler (BioTech, USA).
ISOLATION AND PURIFICATION OF THE RECOMBINANT PROTEIN
Transformed E. coli cells are disrupted and the supernatant obtained after centrifugation is purified by affinity chromatography (most commonly Ni-NTA-based). Ni-NTA-based affinity chromatography uses the specific binding of histidine-tagged proteins to nickel ions immobilised on agarose. The protein extract is applied to the column, where the histidine-tagged proteins are immobilised, while the contaminants pass through the column. The proteins labelled in this way are eluted with imidazole, which competes with the histidine residues for binding to the nickel ions. This results in the clean isolation of the target proteins from the complex mixture. The proteins (destructases) are then used to measure enzymatic activity and to search for suitable inhibitors.
PREPARATION OF IN VITRO BODY BARRIER MODELS
In vitro body barrier models are prepared to test the transfer of various substances, from dietary supplements to potential pharmaceuticals. At the same time, they are more advanced models for the study of physiological processes such as drug resistance or inflammation. In particular, we focus on models of the blood-brain barrier, the intestinal barrier and the cornea of the eye. These models are prepared by culturing multiple cell lines simultaneously on Transwell inserts in different configurations to mimic the in vivo situation as closely as possible. Cells are cultured on the inserts until layers of tight junctions are formed, resulting in an increase in electrical resistance.
Instrumentation: Millicell ERS-2 epithelial volt-ohm meter (Merck Millipore, USA).