Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F21%3A00538239" target="_blank" >RIV/61388963:_____/21:00538239 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1016/j.ab.2020.114002" target="_blank" >https://doi.org/10.1016/j.ab.2020.114002</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ab.2020.114002" target="_blank" >10.1016/j.ab.2020.114002</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA

Popis výsledku v původním jazyce

The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5–60 min) and reduced number of steps, the protocol can be completed within 1–2 h with a minimal cell loss and with excellent reproducibility.

Název v anglickém jazyce

Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA

Popis výsledku anglicky

The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5–60 min) and reduced number of steps, the protocol can be completed within 1–2 h with a minimal cell loss and with excellent reproducibility.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10401 - Organic chemistry

Projekt

<a href="/cs/project/GA17-14791S" target="_blank" >GA17-14791S: Syntetická analoga transportérů nukleosid-trifosfátů: vývoj a aplikace</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytical Biochemistry

ISSN

0003-2697

e-ISSN

1096-0309

Svazek periodika

614

Číslo periodika v rámci svazku

Feb 1

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

114002

Kód UT WoS článku

000603552000005

EID výsledku v databázi Scopus

2-s2.0-85097039369