Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates

Scragg, M. A.; Ferreira, Luiz Fernando Lucas

doi:10.1016/0003-2697(91)90509-r

Cited by 43 publications

(31 citation statements)

References 10 publications

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“…2 A). To quantify the increased colony sizes, the crystal violet in the stained plates was eluted in a 0.1% SDS solution, and the absorbance at 595 nm was measured (20). Consistent with visual inspection, each of the three pairwise combinations showed 4-to 6-fold higher growth than all of the single-transfectant and double-vector control transfectant cells (Fig.…”

Section: The 14q133 Amplicon Contains Three Transcription Factor Genesmentioning

confidence: 54%

Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer

Kendall

Liu

Bakleh

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

206

213

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We used high-resolution array analysis to discover a recurrent lung cancer amplicon located at 14q13.3. Low-level gain of this region was detected in 15% of lung cancer samples, and high-level amplification was detected in an additional 4% of samples. Highlevel focal amplification appears to be specific to lung cancers, because it was not detected in >500 samples of other tumor types. Mapping of the commonly amplified region revealed there are three genes in the core region, all of which encode transcription factors with either established lung developmental function (TTF1/ NKX2-1, NKX2-8) or potential lung developmental function (PAX9). All three genes were overexpressed to varying degrees in amplified samples, although TTF1/NKX2-1 was not expressed in the squamous cancer subtype, consistent with previous reports. Remarkably, overexpression of any pairwise combination of these genes showed pronounced synergy in promoting the proliferation of immortalized human lung epithelial cells. Analysis of human lung cancer cell lines by both RNAi and ectopic overexpression further substantiates an oncogenic role for these transcription factors. These results, taken together with previous reports of oncogenic alterations of transcription factors involved in lung development (p63, CEBPA), suggest genetic alterations that directly interfere with transcriptional networks normally regulating lung development may be a more common feature of lung cancer than previously realized.gene amplification ͉ lung development ͉ lung oncogene ͉ TTF1 NKX2-8 PAX9 ͉ lineage addiction

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Section: The 14q133 Amplicon Contains Three Transcription Factor Genesmentioning

confidence: 54%

Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer

Kendall

Liu

Bakleh

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

206

213

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“…The method to fix cells prior to staining and counting them is yet another source of variation among chemotoaxis studies (16,43,56). We found that methanol and paraformaldehyde methods of cell fixation reduced total counts of migrated cells to 10 ng/ml PDGF-BB by about 40%, whereas 70% ethanol had no effect compared to glutaraldehyde ( The effect of fixation method on counted cells was assessed after chemotaxis to 10 ng/ml PDGF-BB.…”

Section: Optimization Of the Transmembrane Chemotaxis Assaymentioning

confidence: 97%

Fibronectin, Vitronectin, and Collagen I Induce Chemotaxis and Haptotaxis of Human and Rabbit Mesenchymal Stem Cells in a Standardized Transmembrane Assay

Thibault

Hoemann

Buschmann

2007

Stem Cells and Development

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The mesenchymal stem cell (MSC) is a critical element in tissue repair and regeneration. Its ability to differentiate into multiple connective tissue cell types and to self-renew has made it a prime candidate in regenerative medicine strategies. Currently, the environmental cues responsible for in situ recruitment and control of MSC distribution at repair sites are not entirely revealed and in particular the role of extracellular matrix (ECM) proteins as motogenic factors has not been studied. Here we have used a standardized transmembrane chemotaxis assay to assess the chemotactic and haptotactic potential of fibronectin, vitronectin, and collagen type 1 on MSCs from both rabbit and human origin. The use of both cell types was based in part on the widespread use of rabbit models for musculoskeletal-related tissue engineering and repair models and their unknown correspondence to human in terms of MSC migration. The optimized assay yielded a greatly increased chemotactic response toward known factors such as platelet-derived growth factor-BB (PDGF)-BB compared to previous studies. Our primary finding was that all three ECM proteins tested (fibronectin, vitronectin, and collagen I) induced significant motogenic activity, in both soluble and insoluble forms, for both rabbit and human MSCs. These results suggest that ECM proteins could play roles as significant as cytokines in the recruitment of pluripotential repair cells wound and tissue repair sites. Furthermore, designed ECM coatings of scaffolds or implants could provide a new tool to control both cell influx and outflux from the scaffold post-implantation. Finally, the similarity of motogenic behavior of both rabbit and human cells suggests the rabbit is a reliable model for assessing MSC recruitment in repair and regeneration strategies. 489

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“…The synthetic class specific inhibitor, BB94 (Davies et al, 1993b) (Scragg and Ferreira, 1991 Under SF conditions (MDA-MB-231 cells), pepstatin was also ineffective but some apparent reduction in BMD was seen with E64 (84 ± 14%, n = 5) and pepstatin plus E64 (64 ± 13%, n = 4), but this was not statistically significant using the multiple comparisons method. PD 134678-0073 (PD) produced no reduction in BMD with these cells, although this inhibitor had to be used at a lower concentration (10 FtM) because it was cytotoxic at 100 [tM.…”

Section: Inhibition Of Mmpsmentioning

confidence: 99%

Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines

Stonelake

Jones²,

Neoptolemos³

et al. 1997

Br J Cancer

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SummaryThe relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled (Fidler, 1990). Basement membrane degradation (BMD) is brought about by extracellular proteinases, and there is currently interest in inhibitors of these enzymes as potential therapeutic agents. Possible candidates for the proteinases involved in breast cancer invasion are found in each of the four major classes of proteinases, i.e. aspartic, cysteine, serine and matrix metalloproteinases (MMPs).The aspartic proteinase cathepsin D, a lysosomal enzyme, is oversecreted by breast cancer cells in vitro and may be a major proteinase involved in BMD (Briozzo et al, 1988). This view is supported by in vivo experiments in which transfection of cathepsin D cDNA resulted in a higher frequency of metastases in mice (Garcia et al, 1990). Nevertheless, some workers have questioned whether this lysosomal enzyme, which is only active at acid pH, has a role in invasion under physiological conditions (Johnson et al, 1993).The cysteine proteinases, cathepsins B, L and H, are also lysosomal enzymes that are overexpressed in breast cancer (Vasishta et al, 1988;Gabrijelcic et al, 1992). Cathepsin B from normal human liver and from human breast carcinomas has been shown to degrade components of BM at neutral pH as well as at acid pH (Buck et al,

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Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates

Cited by 43 publications

References 10 publications

Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer

Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer

Fibronectin, Vitronectin, and Collagen I Induce Chemotaxis and Haptotaxis of Human and Rabbit Mesenchymal Stem Cells in a Standardized Transmembrane Assay

Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines

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