Biochemistry and Imaging Laboratory - Department of Biochemistry

In the Biochemistry and Imaging Laboratory, the techniques of high-performance liquid chromatography and mass spectrometry are used on a large scale.
Analyzes by these techniques include, but are not limited to:

1. Targeted metabolomic / proteomic analysis:
- amino acids and their derivatives profile (including tryptophan, arginine, citrulline, asymmetric dimethylarginine, monomethylarginine)
- nicotinamide metabolites profile (nicotinamide, methyl-NA, NAD +, nicotinic acid, nicotinamide riboside)
- nucleotide metabolites profile (ATP, ADP, AMP, GTP, GDP, GMP, IMP, inosine, adenosine, xanthine, uric acid, NAD, NADH)
- angiotensin peptide profile (Ang (1-7), Ang I, Ang II, Ang, III, Ang (1-9))
- profile of soluble ecto-enzymes of nucleotide metabolism in plasma (ecto-5'-nucleotidase, adenosine deaminase and others)
- Spatial metabolomics/ proteomics and mass imaging of tissues

2. Non-targeted metabolomic / proteomic analysis:
- Purine and pyrimidine bases, nucleosides, nucleotides (mono-, some di-), and derivatives
- Amino acids
- Organic acids
- Catecholamines
- Microbiota-related compounds (tryptophan metabolites)
- Phosphocholines and other polar lipid groups
- Free fatty acids
- Select steroids, especially sulphonated and polar
- Drugs and their metabolites (confirmed within an epilepsy related study: oxazepam, levetiracetam, pregabalin, carbamazepine, valproic acid)
- Peptides
- Spatial metabolomics/ proteomics and mass imaging of tissues

In Biochemistry and Imaging Laboratory we also offer experimental models for in vitro research on the mechanisms of human diseases and for testing new therapies.

Cell cultures include:
- Vascular endothelial cells (primary: e.g. HAEC, HCAEC, VEC, LEC, cell lines: e.g. HMEC-1, H5V, HUVEC, PIEC)
- Smooth muscle cells (primary VSMC)
- Aortic interstitial cells (primary VIC)
- Cardiomyocytes (cell lines: e.g. H9c2, AC16, HL-1)
- Inflammatory cells (e.g. THP-1, HL-60, Jurkat cell lines)
- Tumor cells (e.g. MCF-1, MDA-MB-231, E0771, 4T1, PANC-1)

The laboratory is equipped with an AxioObserver 7 FL inverted fluorescence microscope with ApoTome.3 module, which allows for live cell imaging under normoxic and hypoxic conditions. The microscope is equipped with two cameras. Axiocam 503 3-megapixel monochrome camera with exceptional sensitivity for low light and live imaging. High sensitivity allows the use of short exposure times, even with weak fluorescent markers, which prevents sample damage. The microscope also allows the observation of histological preparations in transmitted light. The second, ultra-fast 5-megapixel Axiocam 305 color camera uses CMOS Global Shutter technology, which allows high-resolution imaging at high speeds. This makes it possible to obtain up to 36 frames per second in full resolution. The microscope has a system of quick change and individual configuration of up to 64 fluorescent filters. In addition, the microscope is equipped with a Nomarski contrast.

Thanks to AxioObserver 7 FL it is possible to:
- imaging of cellular structures
- survival and automatic measurements of proliferation and migration in cell cultures
- permeability and invasion, proliferation tests.
- analysis of cell adhesion processes
- tracking the moving intra- and extracellular proteins, analysis of their externalization to the surface of the cell membrane
- assessment of cell viability and general condition
- assessment of cell functionality, e.g. analysis of nitric oxide production by the vascular endothelium, analysis of myocyte contractility by assessing intracellular calcium levels
- analysis of histological preparations

The Laboratory also has laminar chambers and cell culture incubators, which provide a wide range of research opportunities and high research throughput.
In addition, advanced cell co-culture models as well as mechanical cell stimulation systems and the Seahorse XFp system are used in the research.

The Seahorse XFp System is an extracellular flux analyzer that represents the Gold Standard in the study of cell metabolism. This system allows for real-time "stress test" of the two main energy production pathways (glycolysis and mitochondrial respiration). The Seahorse XFp analyzer allows to study mammalian cells, yeast, isolated mitochondria or plant cells.

Thanks to Seahorse XFp it is possible to:
- measurement of cellular metabolism
- detection of key changes in cellular metabolism
- experiential and automatic real-time measurement of mitochondrial respiration and glycolysis,
- performing analyzes of eukaryotic cells and mitochondria from a wide spectrum of tissues
- working with small amounts of cells (from 5x103 to 2x105) and mitochondria (1-5 µg)
- assessment of mitochondrial functioning, consumption of respiratory substrates and the general condition of the cell

Analytical Laboratory:
- Orbitrap Exploris 480 mass spectrometer combined with Vanquish liquid chromatography system, Ultimate 3000 nanochromatography system (ThermoFisher) or MALDI(ng) UHR mass imaging source AP- (MassTech)
- MicroTOF-Q2 mass spectrometer (Bruker) combined with liquid chromatography or nanochromatography systems
- Quantum TSQ Vantage EMR mass spectrometer combined with Surveyor chromatography or Ultimate 3000 nanochromatography (ThermoFisher) systems
- Nexera LC 2040 (Shimadzu) and Nexera LC40 (Shimadzu) liquid chromatography systems
- XL 180 biochemistry analyzer (Erba Manheim)

Microscopy and Cell Culture Laboratory:
- AxioObserver 7 FL inverted light and fluorescence microscope with the incubation module for live cell analyses and ApoTome.3 (Carl Zeiss)
- Seahorse XFp analyzer (Agilent)
- Laminar chambers and cell culture incubators