Additional file 8.

CellTiter Glo luminescent cell viability assay validation for three-dimensional cultures. (a) CellTiter Glo assay of day 4 U-87 MG spheroids incubated for 10, 30 or 60 minutes with CellTiter Glo reagents showed close comparability of the luminescent signals. (b) Ultra-low attachment (ULA) plate generated U-87 MG spheroids were imaged and used in a CellTiter Glo luminescent assay on days 3, 4, 5, 6, 8 and 11 post initiation. Spheroid volumes (μm3) and luminescent counts showed a significant positive correlation (Spearman rank). (c) U-87 MG spheroids were initiated in ULA plates at different cell densities (5 × 104, 2.5 × 104, 12.5 × 104, 6.25 × 104 and 3.12 × 104 cell/well). Then, 4 days later, spheroids were imaged and analyzed on a Celigo cytometer. Representative images of three of the cultures are shown. Scale bar: 500 μm. Following imaging, spheroids were subjected to a CellTiter Glo luminescent viability assay (d). For assessment of the relationship between the number of viable cells and the luminescent signals from spheroids, a standard curve was generated. A single cell suspension of U-87 MG was plated into a 96-well plate, over a range of cell densities (105, 104, 103 and 102 cells/well) and immediately subjected to a CellTiter Glo assay alongside the spheroids (e). Volumes (μm3) and the number of viable cells per spheroid determined using the standard curve showed a significant positive correlation (Spearman rank). Values are shown as means ± SD, n = 8.

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Vinci et al. BMC Biology 2012 10:29   doi:10.1186/1741-7007-10-29