Glutamine is involved in the dependency of brain neuron survival on cell plating density in culture

Neuroreport ◽  
1998 ◽  
Vol 9 (10) ◽  
pp. 2353-2357 ◽  
Author(s):  
Masatomo Watanabe ◽  
Yoshihide Ohe ◽  
Kenji Katakai ◽  
Kenji Kabeya ◽  
Yukihito Fukumura ◽  
...  
Keyword(s):  
Brain Neuron ◽  
Neuron Survival ◽  
Plating Density ◽  
Cell Plating

A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential-Sensitive Dye and FLIPR

2009 ◽  
Vol 14 (9) ◽  
pp. 1119-1128 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Qin J. Shan ◽  
Yan T. Lee ◽  
Veronica Soloveva ◽  
Stanley P. Nawoschik ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Channel Blocker ◽  
Fluorescent Imaging ◽  
Fluorescence Signal ◽  
Hcn Channel ◽  
Plating Density ◽  
High Throughput Screening Assay ◽  
Cell Plating

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r2 = 0.56) between the respective IC50s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

scholarly journals Effect of cell-plating density on the steroidogenic activity of human cumulus cells

Reproduction ◽  
1994 ◽  
Vol 101 (3) ◽  
pp. 729-735 ◽  
Author(s):  
S. Bar-Ami ◽  
C. Khoury
Keyword(s):  
Cumulus Cells ◽  
Plating Density ◽  
Cell Plating

ARNT2, a transcription factor for brain neuron survival?

1999 ◽  
Vol 11 (5) ◽  
pp. 1545-1553 ◽  
Author(s):  
Guillaume Drutel ◽  
Anne Héron ◽  
Markus Kathmann ◽  
Claude Gros ◽  
Séverine Macé ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Brain Neuron ◽  
Neuron Survival

scholarly journals Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

1998 ◽  
Vol 9 (11) ◽  
pp. 3119-3131 ◽  
Author(s):  
Polina Goichberg ◽  
Benjamin Geiger
Keyword(s):  
Cell Adhesion ◽  
Long Range ◽  
Myogenic Differentiation ◽  
Growth Arrest ◽  
Muscle Differentiation ◽  
Sarcomeric Proteins ◽  
Plating Density ◽  
Myogenic Induction ◽  
Cell Plating ◽  
Cell Cell

Cell–cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

scholarly journals Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system

Reproduction ◽  
2003 ◽  
pp. 527-538 ◽  
Author(s):  
C Allegrucci ◽  
MG Hunter ◽  
R Webb ◽  
MR Luck
Keyword(s):  
Cell Survival ◽  
Culture System ◽  
Close Contact ◽  
Cell Phenotype ◽  
Serum Free ◽  
Theca Cells ◽  
Plating Density ◽  
Igf I ◽  
Defined Culture ◽  
Cell Plating

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

scholarly journals 297 Distinct Cell Lines: A High-Content Analysis Assay and a Full-Automation Design Solely Using Noncontact Liquid Dispensing

2007 ◽  
Vol 12 (5) ◽  
pp. 318-326 ◽  
Author(s):  
Richard A. Rodriguez ◽  
Jaiver Alfonso ◽  
Christine O'Day ◽  
Yulia Ovechkina ◽  
Courtney Ward
Keyword(s):  
Content Analysis ◽  
Cell Lines ◽  
Performance Metrics ◽  
Automation System ◽  
Data Set ◽  
Plating Density ◽  
High Content Analysis ◽  
Full Automation ◽  
Unique Cell ◽  
Cell Plating

This review assesses the quality of the data acquired over a 13-week period from a High-Content Analysis screening project that used 297 unique cell lines. This article also evaluates the proficiency of a “tipless” (i.e., does not use disposable tips) full-automation design used for this project that prioritizes intralab system mobility and system configuration mutability. The request to assay a large number of cell lines with poorly characterized growth rates led us to devise an MDS PharmaServices, Inc. proprietary algorithm in an effort to select the proper cell plating density for each cell line. The performance metrics include coefficients of variation (CVs) of Controls for the cell plating data and Data Set Mean CVs for assessing replicate propinquity (i.e., how close the replicates are to each other). The performance of the automation system and our algorithm for this project produced data of superior quality.

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