Multi-cellular tumor spheroid assays were preformed essentially as described previously [17 (link)]. 1000 HT29 cells/well were seeded in 96-well round bottomed ultra-low attachment microplates (Corning Costar), centrifuged at 1000 × g for 3 minutes and spheroids formed for 72 hours. Spheroid cell viability after incubation with chemotherapeutic drug plus V158411 for 168 hours was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega).
96 well round bottomed ultra low attachment microplates
Manufactured by Corning
Sourced in United States
The 96-well round-bottomed ultra-low attachment microplates are designed for cell culture applications. The plates feature a round-bottom well shape and a surface that minimizes cell attachment, promoting the formation of 3D cell aggregates or spheroids.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Operetta high content screening system, by PerkinElmer (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Ethd 1, by Thermo Fisher Scientific (2 mentions)
Harmony software, by PerkinElmer (2 mentions)
Ethd 1, by PerkinElmer (2 mentions)
Victor plate reader, by PerkinElmer (1 mentions)
Gefitinib, by Merck Group (1 mentions)
0.45 μm pore filter, by Merck Group (1 mentions)
7 protocols using 96 well round bottomed ultra low attachment microplates
1
Multicellular Tumor Spheroid Viability Assay
2
Cell Proliferation and Viability Assays
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5000 cells per well were seeded in 96-well plates and incubated overnight. Cells were treated with a 10-point titration of compound for 72 or 168 hours. The effect on cell proliferation was determined with sulphorhodamine B (SRB) after fixation with 10% trichloroacetic acid and read on a Victor plate reader (Perkin Elmer). GI50 values were calculated in Microsoft EXCEL using an XLFit software add-in (ID Business Solutions).
2000 cells/well were seeded in 96-well round bottomed ultra-low attachment microplates (Corning Costar), centrifuged at 1000 × g for 3 minutes and spheroids formed for 72 hours. Spheroid cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega).
2000 cells/well were seeded in 96-well round bottomed ultra-low attachment microplates (Corning Costar), centrifuged at 1000 × g for 3 minutes and spheroids formed for 72 hours. Spheroid cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega).
3
Multicellular Tumor Spheroid Radiation Therapy
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Multicellular tumor spheroids were initiated by inoculating 2000 cells in 100 μl growth medium per well into 96‐well round‐bottomed ultra‐low attachment microplates (Corning). At 48 h later, iRGD were added into wells and radiation with the indicated dose was performed after MCTS were cultured with peptides for 4 h. The final concentration of iRGD is 25 μg/mL, except for other concentrations described in some assays. Diameters of the MCTS were measured using ImageJ software every other day. The volume of a spheroid was calculated in accordance with the formula V = (1/6)πd3, where d = diameter.
4
Spheroid and MCTS Generation Protocol
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To generate spheroids, cells suspended in complete medium were seeded at a density of 6 × 103 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning B.V. Life Sciences, Amsterdam, Netherlands). The plates were incubated for 3 days at 37 °C in a humidified atmosphere of 5% CO2. To generate MCTSs containing various cell types, lung cancer cells, and stromal cells (WI38 and HUVECs) were mixed at a 5:5 ratio.
5
Spheroid Cell Death Assay for Lung Cancer
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Non-speci c SiRNA (SiCont) or HYOU1 SiRNA (SiHYOU1) transfected for 24 h or 5, 10, 20, or 40 µM of ge tinib and cisplatin (all from Sigma-Aldrich, St. Louis, MO, USA) treated for 48 h, lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) with or without HUVECs were seeded at a density of 6 × 10 3 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning, NY, USA). After 2 or 3 days, the spheroids cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen, Eugene, OR, USA). EthD-1 is a high-a nity nucleic acid stain that uoresces weakly until bound to DNA, whereupon it emits red uorescence (excitation/ emission maxima ~ 528/617 nm). Spheroids were incubated in 4 µM EthD-1 in complete medium for 30 min in a 37 °C incubator, and images were obtained and the intensity of EthD-1 uorescence measured using the Operetta® High Content Screening System (Perkin Elmer, Waltham, MA, USA). Fluorescent intensity analysis was performed using the Harmony software (Perkin Elmer, Waltham, MA, USA).
6
Assessing Lung Cancer Spheroid Death
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Non-specific siRNA (siCont) or HYOU1 siRNA (siHYOU1)transfected for 24 h or 5, 10, 20, or 40 μM of gefitinib and cisplatin (all from Sigma-Aldrich, USA) treated for 48 h, lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) with or without HUVECs were seeded at a density of 6 × 103 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning). After 2 or 3 days, the spheroids cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen). EthD-1 is a high-affinity nucleic acid stain that fluoresces weakly until bound to DNA, whereupon it emits red fluorescence (excitation/emission maxima ~528/617 nm). Spheroids were incubated in 4 μM EthD-1 in complete medium for 30 min in a 37°C incubator, and images were obtained and the intensity of EthD-1 fluorescence measured using the Operetta® High Content Screening System (Perkin Elmer, USA). Fluorescent intensity analysis was performed using the Harmony software (Perkin Elmer).
7
Lung Cancer Cells Cultivation and Response
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HUVECs, lung cancers (NCI-H460 cells or A549 cells), and lung cancers with HUVECs were cultured in 2D conditions using the same number of cells and amount. Conditioned medium (CM) from cultured HUVECs, lung cancers (NCI-H460 cells or A549 cells), and lung cancers with HUVECs were collected when the cells reached 70%-90% confluence, and passed through a 0.45-μM pore filter (Millipore, USA) to eliminate debris. Lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) were seeded at a density of 6 × 103 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning, USA) with 80 μl of filtered 2D-CM for 48 h.
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