OPTIMALIZÁCIA IN VITRO DIFERENCIÁCIE A AKTIVÁCIE MONOCYTOV PRED ANALÝZOU EXPRESIE GÉNU PLAUR
| Title in English | OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION |
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| Authors | |
| Year of publication | 2022 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | The aim of the work was to optimize the process of isolation, differentiation and activation of monocytes. The aim was to achieve maximum monocyte yields from peripheral blood collection, their high purity and the lowest possible primary cell activation. By selecting the appropriate cytokine concentration and incubation time, we aimed to achieve in-vitro differentiation of monocytes into M1-type macrophages. Subsequently, PLAUR gene expression will be monitored in activated and fully differentiated monocytes from patients with hereditary angioedema, asthma, and rheumatoid arthritis. The PLAUR gene encodes a uPAR protein (CD87) affecting fibrinolysis upon its interaction with urokinase plasminogen activator (uPA), blood coagulation and bradykinin production by binding to FXII, which is activated at the cell surface and modulates cell adhesion and migration through interactions with extracellular matrix proteins. Monocytes were isolated from peripheral blood samples by magnetic separation. The recovered monocytes were differentiated by a combination of the cytokines M-CSF and INF?, which promoted differentiation into M1 macrophages. The purity of the isolated monocytes, their differentiation and activation were determined microscopically and by flow cytometry. Higher monocyte yields were obtained using a doubly concentrated PBS solution with an average monocyte yield of around 150,000 cells per 1 ml of peripheral blood with a purity of over 95%. Low monocyte activation was achieved by using glass tubes and by maintaining a low temperature (< 8 oC) of the cell suspension throughout the isolation process. The most appropriate length of monocyte incubation and concentrations of the cytokines MCSF and INFg were determined by microscopic observation, Monocyte differentiation and activation were determined by flow cytometer measurements based on increased surface expression of the markers CD86, CD38 and CCR7. Thus differentiated and activated monocytes proved to be suitable for further processing. As a result of the performed experiments, an optimized and verified monocyte isolation and activation procedure necessary for accurate analysis of PLAUR gene expression has been established. |
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