OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION
| Název česky | OPTIMALIZACE IN-VIRO DIFERENCIACE A AKTIVACE MONOCYTŮ PŘED ANALÝZOU GENOVÉ EXPRESE PLAUR |
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| Autoři | |
| Rok publikování | 2022 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | The aim of our research is to define PLAUR gene expression after activation of fully differentiated monocytes in patients with hereditary angioedema, asthma bronchiale and rheumatoid arthritis. PLAUR gene codes a multifunctional protein uPAR (CD87) which affects fibrinolysis following an interaction with urokinase plasminogen activator (uPA), influences blood coagulation and bradykinin production through the binding of FXII, which is activated at cell surfaces, and modulates cell adhesivity and migration through the interactions with extracellular matrix proteins. The cornerstone of this project was to optimize the process of isolation, differentiation and activation of monocytes. The effort was to achieve maximum yields of monocytes from the sample of peripheral blood, with purity as highest as possible and the lowest possible primary cell activation. By choosing the appropriate concentration of cytokines and incubation time, we tried to achieve in-vitro differentiation of monocytes into M1 type macrophages. Monocytes were isolated from the samples of peripheral blood using the magnetic separation method. Obtained monocytes were activated by a combination of cytokines M-CSF and INF?, which promoted their differentiation into M1 macrophages. The purity of isolated monocytes, their differentiation and activation were determined using the flow cytometry and optical microscopy. Higher yields of monocytes were achieved using PBS in concentration 2x higher than usually, when the average yield of monocytes was around 150,000 cells from 1 ml of peripheral blood with purity above 95 %. Low activation of monocytes was achieved by using glass tubes and maintaining a low temperature (< 8°C) of the cell suspension throughout the isolation process. Microscopic observation determined the most appropriate length of monocyte incubation and the concentration of MCSF and INFg cytokines. Monocyte differentiation and activation was determined by flow cytometer measurements of surface expression of CD86, CD38 and CCR7 markers. Monocytes differentiated and activated in this way proved to be suitable for further processing. The result of the performed experiments is an optimized and verified protocol for the isolation and activation of monocytes necessary for the precise analysis of the expression of the PLAUR gene. |
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