OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF
MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION

a 2022

OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION

SLANINA, Peter; Julie ŠTÍCHOVÁ; Petra KULÍŠKOVÁ; Lucie BALLONOVÁ; Jan BAROŠ et al.

Základní údaje

Originální název

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

Autoři

SLANINA, Peter; Julie ŠTÍCHOVÁ; Petra KULÍŠKOVÁ; Lucie BALLONOVÁ; Jan BAROŠ; Jiří LITZMAN; Marcela VLKOVÁ; Přemysl SOUČEK; Tomáš FREIBERGER a Roman HAKL

Vydání

MESIA PRAGUE 2022, 2022

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

30102 Immunology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14110/22:00129723

Organizační jednotka

Lékařská fakulta

Klíčová slova česky

Monocyty; PLAUR; izolace; diferenciace

Klíčová slova anglicky

Monocytes; PLAUR; isolation; differentiation

Štítky

rivok

Změněno: 4. 5. 2023 13:57, Mgr. Tereza Miškechová

Anotace

V originále

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 INFγ, 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.

Návaznosti

NU21-05-00438, projekt VaV

Název: Úloha alternativních forem uPAR v rozvoji imunopatologických reakcí

Investor: Ministerstvo zdravotnictví ČR, Úloha alternativních forem uPAR v rozvoji imunopatologických reakcí, Podprogram 1 - standardní

Citovat

SLANINA, Peter; Julie ŠTÍCHOVÁ; Petra KULÍŠKOVÁ; Lucie BALLONOVÁ; Jan BAROŠ; Jiří LITZMAN; Marcela VLKOVÁ; Přemysl SOUČEK; Tomáš FREIBERGER a Roman HAKL. OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION. In MESIA PRAGUE 2022. 2022.

@proceedings{2244878,
   author = {Slanina, Peter and Štíchová, Julie and Kulíšková, Petra and Ballonová, Lucie and Baroš, Jan and Litzman, Jiří and Vlková, Marcela and Souček, Přemysl and Freiberger, Tomáš and Hakl, Roman},
   booktitle = {MESIA PRAGUE 2022},
   keywords = {Monocytes; PLAUR; isolation; differentiation},
   language = {eng},
   title = {OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION},
   year = {2022}
}

TY  - CONF
ID  - 2244878
AU  - Slanina, Peter - Štíchová, Julie - Kulíšková, Petra - Ballonová, Lucie - Baroš, Jan - Litzman, Jiří - Vlková, Marcela - Souček, Přemysl - Freiberger, Tomáš - Hakl, Roman
PY  - 2022
TI  - OPTIMIZATION OF IN-VITRO DIFFERENTIATION AND ACTIVATION OF MONOCYTES BEFORE THE ANALYSIS OF PLAUR GENE EXPRESSION
KW  - Monocytes
KW  - PLAUR
KW  - isolation
KW  - differentiation
N2  - 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 INFγ, 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.
ER  -

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