scholarly journals A Modified Limiting Dilution Method for Monoclonal Stable Cell Line Selection Using a Real-Time Fluorescence Imaging System: A Practical Workflow and Advanced Applications

2021 ◽  
Vol 4 (1) ◽  
pp. 16
Author(s):  
Mingyu Ye ◽  
Martina Wilhelm ◽  
Ivaylo Gentschev ◽  
Aladár Szalay
Keyword(s):  
Cell Line ◽  
Real Time ◽  
Cell Lines ◽  
Imaging System ◽  
Stable Cell Line ◽  
Dilution Method ◽  
Fluorescence Imaging System ◽  
Line Selection ◽  
Cell Line Selection ◽  
Limiting Dilution

Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte®S3.

scholarly journals A Place for High-Throughput Electrophysiology in Cardiac Safety: Screening hERG Cell Lines and Novel Compounds with the Ion Works HTTM System

2005 ◽  
Vol 10 (8) ◽  
pp. 832-840 ◽  
Author(s):  
Heather Guthrie ◽  
Frederick S. Livingston ◽  
Ueli Gubler ◽  
Ralph Garippa
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Torsades De Pointes ◽  
Delayed Rectifier ◽  
Drug Candidates ◽  
Delayed Rectifier Potassium Channel ◽  
A Cell ◽  
Line Selection ◽  
Herg Channels ◽  
Cell Line Selection

Several commercially available pharmaceutical compounds have been shown to block the I Krcurrent of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QTinterval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for I Krblock to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go– related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of I Kr block. One strategy to identify compounds withh ERGliability is to monitor hERGcurrent inhibition using electrophysiology techniques. The authors describe the Ion Works HT ™instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT ™system, provide a powerful means of assessinghERGactive compounds early in the drug discovery pipeline.

scholarly journals Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Rileen Sinha ◽  
Andrew G. Winer ◽  
Michael Chevinsky ◽  
Christopher Jakubowski ◽  
Ying-Bei Chen ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Renal Cancer ◽  
Cancer Cell Lines ◽  
Renal Cancer Cell ◽  
Line Selection ◽  
Cell Line Selection

scholarly journals Does earlier use of productivity enhancers during cell line selection lead to the identification of more productive cell lines?

2011 ◽  
Vol 5 (S8) ◽  
Author(s):  
Alison J Porter ◽  
Atul Mohindra ◽  
Juana Maria Porter ◽  
Andrew J Racher
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Line Selection ◽  
Cell Line Selection

Guide for Selection of Relevant Cell Lines During the Evaluation of new Anti-Cancer Compounds

2018 ◽  
Vol 18 (8) ◽  
pp. 1072-1081
Author(s):  
Angel J. Ruiz-Moreno ◽  
Patricia Torres-Barrera ◽  
Mireya Velázquez-Paniagua ◽  
Alexander Dömling ◽  
Marco A. Velasco-Velázquez
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Activity Analysis ◽  
Anti Cancer ◽  
Line Selection ◽  
Cell Line Selection ◽  
Selection Of

Background: Human cancer cell lines are valuable models for anti-cancer drug development. Although all cancer cells share common biological features, each cancer cell line has unique genotypic/ phenotypic characteristics that affect drug response. Thus, the information obtained with a specific cancer cell line cannot be easily extrapolated to other cancer cells. Consequently, cell line selection during experimental design is critical for providing proper and clinically relevant structure-activity analysis. Methods: Herein, we critically review the use of cancer cell lines as tools for activity analysis by comparing two different scenarios: i) the use of multiple cancer cell lines, with the NCI-60 Program as the most representative example; and, ii) the selection of a single cell line with specific biological characteristics that match the rationale of compound design. Results: Considering that most laboratories evaluate the activity of new compounds using few cell lines, we provide a systematic strategy for selection based on the expression levels and genetic status of the target and the effectiveness of target inhibition or silencing. We exemplify the use of public databases for data retrieval and analysis as well as the critical comparison of such information with published results. Conclusion: This approach refines cell line selection, avoiding the perpetuation of published poor selection and enhancing the relevance of the results.

scholarly journals A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

mAbs ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 416-430 ◽  
Author(s):  
Gargi Roy ◽  
Tom Martin ◽  
Arnita Barnes ◽  
Jihong Wang ◽  
Rod Brian Jimenez ◽  
...  
Keyword(s):  
Cell Line ◽  
Stable Cell Line ◽  
Gene Design ◽  
Line Selection ◽  
Cell Line Selection

Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells

2006 ◽  
Vol 96 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Louise M. Barnes ◽  
Catherine M. Bentley ◽  
Nicola Moy ◽  
Alan J. Dickson
Keyword(s):  
Cell Line ◽  
Molecular Analysis ◽  
Myeloma Cells ◽  
Line Selection ◽  
Cell Line Selection

Rescue of defective SV40 from a transformed mouse 3T3 cell line: Selection of a specific defective

Virology ◽  
1974 ◽  
Vol 60 (2) ◽  
pp. 342-352 ◽  
Author(s):  
Kunito Yoshiike ◽  
Akemi Furuno ◽  
Seijiro Uchida
Keyword(s):  
Cell Line ◽  
Line Selection ◽  
Cell Line Selection ◽  
Selection Of

Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4547-4547
Author(s):  
Huanling Zhu ◽  
Ting Liu ◽  
Yongqian Jia
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Sensitive Cell ◽  
Imatinib Resistance ◽  
Low Expression

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

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