The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs.

• MeSH
• dextrany metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• isochinoliny metabolismus   MeSH
• kultivované buňky MeSH
• mezerový spoj metabolismus   ultrastruktura   MeSH
• mezibuněčná komunikace * MeSH
• myši MeSH
• rhodaminy metabolismus   MeSH
• Sertoliho buňky MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

• MeSH
• fluorescenční mikroskopie metody   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• mezerový spoj * MeSH
• mezibuněčná komunikace * MeSH
• molekulární zobrazování metody   MeSH
• počet buněk * MeSH
• počítačové zpracování obrazu metody   MeSH
• viabilita buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biologický mechanismus účinku modelového tumor promotoru Ethylenglykolu na mezibuněčné komunikace u savčích somatických buněk in vitro metodami metabolic cooperation assay a Scrape loading,za účelem interpretace procesu karcinogeneze.

• MeSH
• ethylenglykol MeSH
• mezibuněčné spoje antagonisté a inhibitory   MeSH
• nádory etiologie   MeSH
• vrozené vady etiologie   MeSH

• Konspekt
• Biologické vědy
• NLK Obory
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Řešení fenoménu dočasné inhibice mezibuněčných komunikací a odolnosti savčích somatických buněk in vitro k opakované expozici tumor promotorem metodami Metabolic cooperation assay a Scrape loading za účelem interpretace procesu karcinogeneze.

• MeSH
• biologické modely MeSH
• ethylenglykoly MeSH
• mezibuněčné spoje MeSH
• nádorová transformace buněk MeSH

• Konspekt
• Biologické vědy
• NLK Obory
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

PURPOSE: To assess whether human papillomavirus (HPV) DNA detection in cervical cancer specimens, or antibodies to selected HPV 16 peptides are predictors of tumor recurrence and long-term survival in patients with squamous cell invasive cervical cancer. SUBJECTS AND METHODS: Four hundred seventy-one cases included in two population-based case-control studies underwent follow-up evaluation. The survival and cause of death were ascertained for 410 cases (87%), with a median follow-up time of 4.6 years after diagnosis. HPV DNA was assessed using an L1 polymerase chain reaction (PCR)-based system and Southern hybridization (SH) on scraped cytologic specimens or biopsies. HPV 16 antibodies to E2, L2, and E7 peptides were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Clinical stage was the only independent prognostic factor for recurrence or survival. Although seropositivity to HPV 16 E7/3 peptide predicted a twofold excess risk of mortality (adjusted hazards ratio [HRa] = 2.0; 95% confidence interval [CI], 1.2 to 3.3), the association was restricted to stage I (HRa = 6.6; 95% CI, 1.2 to 37.6) and II (HRa = 5.9; 95% CI, 2.1 to 16.5) patients. The presence of HPV DNA (HRa = 0.9; 95% CI, 0.5 to 1.5), different estimates of the HPV viral load and the HPV type identified were not predictors of tumor recurrence or survival. CONCLUSION: The presence of antibodies to HPV 16 E7 proteins is of prognostic value in early-stage cervical cancer. Our results provide strong evidence that detection and typing of HPV DNA in cervical cells or tissues is not a prognostic factor for recurrence or survival.

• MeSH
• analýza přežití MeSH
• DNA virů * izolace a purifikace   MeSH
• dospělí MeSH
• ELISA MeSH
• invazivní růst nádoru MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory děložního čípku * chemie   mortalita   patologie   virologie   MeSH
• odds ratio MeSH
• Papillomaviridae * genetika   imunologie   MeSH
• polymerázová řetězová reakce MeSH
• prediktivní hodnota testů MeSH
• prognóza MeSH
• protilátky virové * krev   MeSH
• riziko MeSH
• spinocelulární karcinom * chemie   mortalita   patologie   virologie   MeSH
• staging nádorů MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH