For spheroid generation, 200 μl/well of cell suspensions at optimized densities (0.5 × 104 cells/ml for U-87 MG, KNS42 and LICR-LON-HN4; 1.5 × 104 for MDA-MB-231 P and M variants) were dispensed into ULA 96-well round-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) using a multichannel pipette. Plates were incubated for 4 days at 37°C, 5% CO2, 95% humidity. Where indicated, optimal three-dimensional structures were achieved by addition of 2.5% Matrigel as previously described [52 (link)]. Fully automated image analysis of tumor spheroids was carried out on a Celigo cytometer (Cyntellect Inc, San Diego, CA, USA; http://www.cyntellect.com/content/products/celigo/index.html ), which is equipped with a 4-megapixel CCD camera with an F-theta scan lens (1 μm/pixel, 0.25 NA, 3.5 ×). Images were acquired and analyzed by using the Colony Counting Embryoid Body application with the option to scan 1/16 field of view/well. The width of 1 field of view (FOV) is 975 pixels/2,057 μm and image file size is 0.41 MB. Further technical details of imaging parameters are shown in Additional file 15 .
For the lower throughput method, images were captured using an inverted microscope (Olympus IX 70, (Olympus Microscopy, Southend-on-SeaEssex, UK) equipped with a CCD camera (QImaging, Surrey, BC, Canada and imported into Image-Pro Plus Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA;http://www.mediacy.com/index.aspx?page=IPP ) and by either using macros or manually, multiparametric analysis was performed. In both cases, the radius of each tumor spheroid was used to calculate the volume (μm3): V = 4/3 π r3.
For comparison with our system, U-87 MG cells were also used to generate spheroids using conventional methods as follows: (1) agar-coated 96-well flat-bottomed plates (BD Biosciences, Oxford, England) as previously described [26 (link)] (0.5 × 104 cells/ml as in the ULA 96-well round-bottomed plates); (2) poly-Hema-coated 24-well plates (2 × 105 cells/ml, 1 ml/well). A stock solution of 6 mg/ml poly-Hema (Sigma-Aldrich Company Ltd., Dorset, England) in 95% ethanol was prepared and diluted 1:10 in ethanol. A total of 100 μl/well was dispensed and left to dry before cell addition; (3) RCCS (105 cells/ml, 10 ml/disposable vessel).
In all cases, an inverted microscope was used for image analysis as described above.
For the lower throughput method, images were captured using an inverted microscope (Olympus IX 70, (Olympus Microscopy, Southend-on-SeaEssex, UK) equipped with a CCD camera (QImaging, Surrey, BC, Canada and imported into Image-Pro Plus Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA;
For comparison with our system, U-87 MG cells were also used to generate spheroids using conventional methods as follows: (1) agar-coated 96-well flat-bottomed plates (BD Biosciences, Oxford, England) as previously described [26 (link)] (0.5 × 104 cells/ml as in the ULA 96-well round-bottomed plates); (2) poly-Hema-coated 24-well plates (2 × 105 cells/ml, 1 ml/well). A stock solution of 6 mg/ml poly-Hema (Sigma-Aldrich Company Ltd., Dorset, England) in 95% ethanol was prepared and diluted 1:10 in ethanol. A total of 100 μl/well was dispensed and left to dry before cell addition; (3) RCCS (105 cells/ml, 10 ml/disposable vessel).
In all cases, an inverted microscope was used for image analysis as described above.
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