1. Laboratory of Cellular Biology
2. Laboratory of Molecular Biology
3. Laboratory of Flow Citometry and Histology
4. Dermatological Research Group of the Hungarian Academy of Sciences, University of     Szeged
5. Allergy Research Laboratory

1. Laboratory of Cellular Biology

Cell separation (keratinocyte, melanocyte, fibroblast, lymphocyte)

Several cell types are separated in our laboratory. We gain mononuclear cells and granulocytes from blood, epidermal cells (keratinocytes and melanocytes) and dermal fibroblasts from human skin. These separated cells are directly examined or cultured in adequate environment, and the cell culture is used for our experiments.

Cell culturing (keratinocyte, melanocyte, fibroblast, lymphocyte, SZ95 (sebocyte cell line), HaCaT (keratinocyte cell line), PK (vaginal epithelial cell line), SK MEL23 (melanocyte cell line), HT-MEL (amelanotic melanoma cell line), RBL (rat basophil leukaemia cell line), MEWO (melanin containing cell line)

The separated cells are cultured under huminifizied culture conditions (37°C and 5% CO2-content). In our laboratory, melanocytes, keratinocytes, endothelial cells, fibroblasts, several cell lines and mononuclear cells are cultured.

MTT assay (cell proliferation)

This method is used to examine of cell viability and proliferation. The metabolically active cells get on the tetrazolium-bromide by endocytosis and they convert it for formazane crystals. These crystals are solved in HCl-containing isopropil-alcohol and SDS. The color-intensity of this solution commensurable to the viability, respectively to the number of the cells

Immunhistochemistry

This method is used to the demonstration of several intracellular and cell surface molecules. We examine frozen and paraffin embedded blocks, which are prepared from healthy and pathological skin.

Immuncytochemistry

This method is used to the demonstration of several intracellular and cell surface molecules. The histochemical reaction is performed on special sections, which are prepared from cultured cells by cytocentrifuge.

Staining cells for Flow Cytometry

Surface and intracellular molecules are stained with fluorescent conjugated monoclonal antibodies.

Measurement of NO with Greiss method

We investigate the amount of NO released due to different stimulations.

Elaboration of an ex vivo skin model

We aimed to elaborate an ex vivo skin model whom viability will be longer than the other models in the technical literature. In the future several biological processes (e. g. skin senescence) could be investigated by this model.

L-DOPA staining

This method is used to the detection of the activity of tyrosinase in melanin-producer cells.

UVB-Irradiation

It’s proven that UVB-light has a plenty of physiological effects. In our laboratory we perform numerous experiments in which we examine the effect of UVB light on differet cell types.

Direct measure of melanin

The melanin content of melanocytes and melanoma cells could be measure directly by solving of the cells in NaOH. We examine the effect of several substances and the UVB irradiation on melanin content of the cells by detection of absorbances.

Candida killing

This method is able to investigate the killing and fagociting capacity of the cells. We used it mainly for the examination of granulocytes, buti t can be applied for other cell types.

Chemotaxis

Principally the granulocyte chemotactin recognition and migration could be examined with this method, but it is useful for other cell types, too.

Transfection

Method for the production cell line which expresses determined gene fragment stably or transiently.

LTT (Lymphocyte transformation test)

This method is used for the detection of drug allergy. Lymphocytes are separated from the peripheral blood and are incubated with different concentrations of the examined drugs. The viability and the proliferation of the cells is detected by MTT assay. The severity of the drug allergy can be characterized with the amount of activated lymphocytes.

2. Laboratory of Molecular Biology

PCR (polimerase chain reaction)

RNA is isolated with Trizol reagent from cell culture and tissue samples and transcripted with reverse transcriptase enzime into cDNA. The amount of mRNA is measured with real-time, reverse transcriptase PCR (Q-RT-PCR) method. In some cases comparision analysis between different samples is carried out with semi-quantitative PCR technique. Primer constructing: gene specific, short oligonucleotides used for semi-quantitative PCR method.

Gel-electrophoresis

Used for detection, documentation and densitometry of the products of semi-quantitative PCR reactions and PCR amplifications carried out on genomic DNA.

in vitro DNS techniques

Insertion of DNS fragments into vectors and their amplification by competent E. Coli cells. Transient transfection and stabile transformation into the human cells and cell cultures.

Genomic investigations

After documentation of the clinical parameters with detailed questionnaires, blood and skin samples are obtained from the patients and genomic investigations have been carried out such as PCR-RFLP, Assay-On-Demand, Assay-by-Design kits and sequencing analyses. The genetic background of the following diseases are examined: porphyries, acne vulgaris, acne inversa, melanoma, atopic dermatitis, vitiligo, venous leg ulcer and allergic rhinitis.

Enzyme-linked immunosorbent assay (ELISA)

ELISA assay is used to detect the substances that are produced and secreted (e.g. growth factors, cytokines) into their supernatant by cells, as well as substances from serum.

Western blot

Proteins located in cells and/or on their surface can be detected by this sensitive method based on antigen-antibody reaction.

3. Laboratory of Flow Citometry and Histology

Flow cytometry

phenotype of immune cells, detection of cytokins, cell proliferation assays

Tissue fax

analysis and classification of cells by both conventional transmission light microscopy and multi-channel fluoescence microscopy, combining detailed morphologic information with multi-channel flow cytometry

4. Dermatological Research Group of the Hungarian Academy of Sciences, University of Szeged

See at Laboratory of Cellular Biology and Laboratory of Molecular Biology.

5. Allergy Research Laboratory

PCR (polimerase chain reaction)

RNA is isolated with Trizol reagent from cell culture and tissue samples and transcripted with reverse transcriptase enzime into cDNA. The amount of mRNA is measured with real-time, reverse transcriptase PCR (Q-RT-PCR) method. In some cases comparision analysis between different samples is carried out with semi-quantitative PCR technique. Primer constructing: gene specific, short oligonucleotides used for semi-quantitative PCR method.

Cell separation

Several cell types are separated in our laboratory. We gain mononuclear cells and granulocytes from blood, epidermal cells (keratinocytes and melanocytes) and dermal fibroblasts from human skin. These separated cells are directly examined or cultured in adequate environment, and the cell culture is used for our experiments.

Cell culturing:

The separated cells are cultured under huminifizied culture conditions (37°C and 5% CO2-content). In our laboratory, melanocytes, keratinocytes, endothelial cells, fibroblasts, several cell lines and mononuclear cells are cultured.