Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates

Abstract

Summary This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Crosssections of the colon (HT29, SW620, SWI 116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line had reached confluence, but each of them displayed a specific pattern of cell stacking which ranged from two to ten layers. Postconfluent HT29 cells displayed morphologic features suggestive of some degree of enterocytic differentiation. Growth and cytotoxicity could be studied reliably and reproducibly in this system with the sulforhodamine B protein assay. Against HT29 postconfluent cultures, the EC50's (drug concentrations producing absorbance readings 50% lower than those of non-treated wells) of 5-fluorouracil and of the ether lipid, hexadecylphosphocholine, were 1 mm and 50 gM respectively. The Mueller-Klieser, 1987 andSutherland, 1988 Miedema, 1965;Skehan et al., 1986;Pelletier et al., 1990) or plateau-phase (Twentyman, 1976;Drewinko et al., 1981) tumour cell cultures in order to increase the degree with which in vivo cell-cell interactions, growth physiology and microenvironment conditions are simulated in vitro. It has been suggested that results generated with these systems more closely resemble those obtained with spheroids and with in vivo models than those obtained with monolayer cell cultures.

Expression of protein tyrosine phosphatase alpha (RPTPα) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo

“…Parental MCF7 cells (&), a vector (control) transfected MCF7 cell clone (*), and three RPTPa-expressing clones (^: a-4;~: a-6, ! : a-9) were seeded at equal initial densities, and growth measured using the Sulphorodamine B dye uptake method (Pizao et al, 1992). The t=0 timepoint corresponds to 18 h after plating.…”

“…Cells were then washed with PBS, and incubated for 30 min with 0.4% Sulphorodamine B in 1% acetic acid (100 ml/well). After three washes in 1% acetic acid, the dye was dissolved in 10 mM Tris pH 10.5 (100 ml/well) and spectrophotometrically evaluated at 492 nm (Pizao et al, 1992).…”

Intercellular contact-dependent survival of human A549, NCI-H596 and NCI-H520 non-small cell lung carcinoma cell lines

“…Pizao et al (16) have established that the SRB-staining technique is suitable to assess The cells attached to the plate were lysed and then centrifuged for 15 min at 14,000 rpm. Subsequently, both the supernatant and the remaining pellet containing larger DNA fragments were counted.…”

The Role of RPTP-Alpha-Like Protein Tyrosine Phosphatases in Mammary Tumorigenesis

“…Differences were considered significant at p<0.05. acetic acid (100 |il/well). After three washes in 1% acetic acid, the dye was dissolved in 10 mM Tris-HCl pH 10.5 (100 pil/well) and spectrophotometrically evaluated at 492 nm [41].…”

“…3b), presumably due to the specific dephosphorylation of the negative regulatory site Y527 by RPTPa. Analysis of the in vitro growth properties of the clones by Sulphorodamine B proliferation assay [41] revealed that the growth rate of the four clones was significantly decreased by RPTPa expression, in comparison with that of parental or empty vector-transfected cells (fig 3c).…”