Quantification of cells cultured on 96-well plates

Kueng, Willy; Silber, E.; Eppenberger, Urs

doi:10.1016/0003-2697(89)90710-0

Cited by 607 publications

(379 citation statements)

References 4 publications

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“…After 24 h from seeding (day 0) and on days 2 and 4, the medium was replaced (in quadruplicate) with complete medium containing NAMI-A, with complete medium alone (positive control), or with starvation SFM (negative control). Cell number was estimated on day 6 by the colorimetric method of Kueng et al (1989). Briefly, cells were fixed for 15 min at room temperature with 2.5% glutaraldehyde, stained for 20 min with 0.1% crystal violet in 20% methanol, solubilised with 10% acetate, and read at 595 nm (Microplate Reader 3550, Bio-Rad Laboratories, Richmond, CA, USA).…”

Section: Proliferation Assaymentioning

confidence: 99%

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A

et al. 2002

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NAMI-A is a ruthenium-based compound with selective anti-metastasis activity in experimental models of solid tumours. We studied whether this activity was dependent on anti-angiogenic ability of NAMI-A. We thus investigated its in vitro effects on endothelial cell functions necessary for angiogenesis to develop, as well as its in vivo effects in the chick embryo chorioallantoic membrane model. Endothelial cell proliferation, chemotaxis, and secretion of the matrix-degrading enzyme metalloproteinase-2 were inhibited by NAMI-A in a dose-dependent manner, and without morphologic signs of cell apoptosis or necrosis. Lastly, NAMI-A displayed a dose-dependent in vivo anti-angiogenic activity in the chorioallantoic membrane model. These data suggest that the anti-angiogenic activity of NAMI-A can contribute to its anti-metastatic efficacy in mice bearing malignant solid tumours.

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Section: Proliferation Assaymentioning

confidence: 99%

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A

et al. 2002

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“…The cells were then fixed with acetic-alcohol fixative (2% acetic acid in 95°ethanol) for 10 min and stained with 0.1% Crystal violet for 30 min. 28 The wells were washed 3 times in water for 10 min and adhering cells were dried. The dye was dissolved for 5 min in 1 ml of 10% acetic acid and the optical density (OD) was measured at 588-nm.…”

Section: Cell Attachment Assaymentioning

confidence: 99%

Inhibition of tumor growth and metastatic spreading by overexpression of inter‐alpha‐trypsin inhibitor family chains

Paris

Sesboüé

Delpech³

et al. 2002

Intl Journal of Cancer

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The inter-␣ trypsin inhibitor (ITI) family is a group of proteins built up from different combinations of 1 light chain (ITI-L) and 3 highly homologous heavy chains (ITI-H1, -H2 and -H3). To investigate a potential role of the ITI family chains in cancer and metastasis spreading, we engineered human H460M cell lines expressing both the green fluorescent protein (GFP) and one of these chains. These clones were subcutaneously injected in athymic nude mice, and lung metastasis number and primary tumor weight were determined after 28 days. Expression of the ITI-L chain considerably decreased tumor weight and fluorescent lung metastasis number. ITI-H1 and ITI-H3 chain expression induced a significant decrease of metastasis number, whereas no decrease of tumor weight could be detected. Key words: inter-␣-trypsin inhibitor; green fluorescent protein; spontaneous lung metastases; primary tumorInhibition of the metastatic process represents an important challenge in cancer treatment, and potential therapeutic approaches include the inhibition of matrix metalloproteinases, 1 of the vascular endothelial cell growth factor (VEGF) pathway 2 or the activation of metastasis suppressor genes. 3 Although most of the molecules involved in the metastatic process have not been identified, it is now established that alteration of the extracellular matrix (ECM) plays an important role in cell migration and metastasis. 4 The major ECM component, hyaluronic acid (HA), is organized as a complex network of macromolecules involving hyaluronic acidbinding proteins (HABP) 5 and the ITI family. The most of ITI members are composed of variable associations of light (ITI-L) and heavy chains (ITI-H1, -H2, -H3), encoded by 4 different genes on 3 chromosomes, 6 -9 giving rise to a series of proteins covalently linked by a glycosaminoglycan 10 and differing in their chain composition. 11 Although the binding of ITI to HA is known for a long time, 12 involvement of heavy chains in this linkage was only recently demonstrated 13-15 together with a major role in the ECM stability of the mouse cumulus oocyte complex. 16 The ITI-L chain mRNA encodes 2 tandemly arranged proteins that separate after proteolytic cleavage: the ␣ 1 -microglobulin (␣ 1 m) that belongs to the lipocalin superfamily 17 and the bikunin that contains 2 Kunitz inhibitory domains responsible for the protease inhibitory activity of ITI molecules. 18 Different studies have suggested a potential antimetastatic effect of bikunin: (i) it inhibits in vitro tumor cell receptor-bound plasmin 19 and the invasiveness of malignant cells; 20,21 and (ii) injections of human purified bikunin efficiently reduced the number of metastases produced by the murine 3LL cell line. 22 We have therefore analyzed in the present study the antimetastatic effect of the different ITI chains using a model of fluorescent spontaneous lung metastases. 23 MATERIAL AND METHODS Cells lines and cultureThe H460M cell line, initially derived from the parental NCI-H460 cell line established from a large-cell carcinoma o...

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“…Dye was dissolved in 10% acetic acid and absorbance measured at 595 nm. 36 The percentage of adherent cells was calculated with respect to the absorbance by the starting population (1 Â 10 5 cells), which was taken as 100%. Mean values 6 SD of triplicates are shown.…”

Section: Adhesion Assaymentioning

confidence: 99%

Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin‐3 and supports cell migration

Paret

Bourouba

et al. 2005

Intl Journal of Cancer

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C4.4A is a member of the Ly6 family, with low homology to uPAR. It has been detected mainly on metastasizing carcinoma cells and proposed to be involved in wound healing. So far, C4.4A has been observed as an orphan receptor, and its functional activity has not been explored. Using recombinant rat C4.4A (rrC4.4A) made in a eukaryotic expression system, we demonstrate by immunohistology that C4.4A ligands are strongly expressed in tissues adjacent to squamous epithelia of, e.g., tongue and esophagus, the expression pattern partly overlapping with laminin (LN) and complementing the C4.4A expression that is found predominantly on the basal layers of squamous epithelium. ELISA screening of several components of the extracellular matrix revealed selective binding of rrC4.4A to LN1 and LN5 and that transfection of the BSp73AS tumor line with C4.4A cDNA (BSp73AS-1B1) promoted LN1 and LN5 binding. Binding of BSp73AS-1B1 to LN5 and, less markedly, LN1 induced spreading, lamellipodia formation and migration. C4.4A also associates with galectin-3 in nontransformed tissues and tumor lines. There is evidence that the association of C4.4A with galectin-3 influences LN adhesion. C4.4A was described originally as a metastasis-associated molecule. Our findings that LN1 and LN5 are C4.4A ligands, that galectin-3 associates with C4.4A and that C4.4A ligand binding confers a migratory phenotype are well in line with the supposed metastasis association. ' 2005 Wiley-Liss, Inc.Key words: C4.4A; laminin; galectin; cell migration LNs are adhesive glycoproteins found in the basement membrane. Fifteen LN isoforms have been identified. 1 LN isoforms are tissue-specific and expressed in a developmentally regulated pattern. 2,3 Cell adhesion to LN plays an important role in maintaining normal tissue organization. LNs promote cell adhesion and migration, 4 are involved in the control of cell proliferation and gene expression 5 and stimulate neurite outgrowth, 6 the multitude of functions being brought about by a large array of receptors. 7 The most prominent LN receptors are integrins, but distinct receptors have also been described, e.g., a-dystroglycan, syndecans, the 67 kDa LN receptor and galectins. 7,8 a-Dystroglycan provides a link between the basement membrane and the dystrophin-actin cytoskeleton. 9 Its binding to LN is Ca 2þ -dependent 10 and requires carbohydrate moieties. 11 Binding of syndecans 2 and 4 to LN5 induces expression of MMP-1. 12 Moreover, syndecan 1 interacts with unprocessed LN5 in migrating keratinocytes, and this interaction depends on the presence of the glycosaminoglycan chain. 13 Galectins belong to the family of galactose-specific lectins and bind the lactosamine-type sugars on LN1. 8 However, at high concentrations, galectins 3 and 1 can downregulate adhesion to LN, probably by blocking the cellular attachment sites. 14,15 The 67 kDa LN receptor can act in concert with a6b4 to promote cell adhesion to LN1. 16 It is also involved in the degradation of LN1 and the release of motility fragments. 17 The GPI-ancho...

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Quantification of cells cultured on 96-well plates

Cited by 607 publications

References 4 publications

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A

Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A

Inhibition of tumor growth and metastatic spreading by overexpression of inter‐alpha‐trypsin inhibitor family chains

Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin‐3 and supports cell migration

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