H9-hESCGFP+ colonies were dissociated into single cells using 10–15-minute incubation with Accutase. Predetermined numbers of single cell dissociated hESCs (6 × 106 cells) were seeded onto each well of Aggrewell™ 800 plates (StemCell Technologies) in Aggrewell media (StemCell Technologies) supplemented with Y27632 (10 μM). The single cell dissociated cells were forcibly aggregated onto the microwells by centrifugation at 100 ×g for 3 minutes. The single cell dissociated hESCs formed clusters of uniform size within the microwells after overnight incubation. These clusters of hESCs would be referred to as spin-embryoid bodies (spin-EBs). The spin-EBs were harvested after 24 hours and transferred to ultralow-attachment six-well culture plates (Corning) in Aggrewell media for 5 days.
Ultralow attachment six well culture plates
Manufactured by Corning
Sourced in United States
Ultralow attachment six-well culture plates are designed for cell culture applications that require a non-adhesive surface. These plates are made of clear polystyrene and have a hydrophilic, low-attachment coating that prevents cell attachment and promotes the formation of cell aggregates or spheroids.
Automatically generated - may contain errors
4 protocols using ultralow attachment six well culture plates
1
Uniform Spin-Embryoid Body Formation
2
Spheroid Culture Assay for Cancer Cells
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Spheroid culture assay was performed as previously described [10 (link)]. Cells were plated at a density of 2500 cells/well in ultra-low attachment six-well culture plates (Cat# 3471, Corning, NY, USA) in DMEM/F12 spheroid media (Cat# 21331020, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and media supplemented with B27 (Cat#17504044, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and N-2 (Cat# 17502001, Gibco, ThermoFisher Scientific, Waltham, MA, USA). Spheres were counted on day 7 and collected on day 10. Images were captured using a PrimoVert Zeiss light microscope (Carl Zeiss Microscopy, White Plains, NY, USA). The experiment was performed in triplicate and the sphere numbers were counted. Images were analyzed to assess the sphere area using Zeiss ZEN Blue lite software (Carl Zeiss Microscopy, White Plains, NY, USA) (10 spheres per group, when possible; if the number was lower, all spheres were measured).
3
Etomoxir Inhibits Tumor Sphere Formation
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H460 cells (2,500–5,000 cells/well) or LNCaP cells (10,000 cells per well) were seeded in ultralow attachment six-well culture plates (Corning, St. Louis, MO, USA) in stem cell media (DMEM/F12 media supplemented with B27 and N2). Additionally, for LNCaP spheroid culture, 0.5 mL media with recombinant EGF (20 ng/mL) and FGF (10 ng/mL) were added every 72 h. At day 7 (or as indicated), spheres were irradiated (2.5 Gy as a single fraction dose) using an RS 2000 Biological Irradiator (Rad Source Technologies, Buford, Georgia 30518, USA). Spheres were immediately treated with etomoxir (experiment day 0) and every 48 h thereafter. At the end, sphere numbers was counted and spheres’ area was measured using AxioVision Release 4.7 software. A brief summary of the experimental scheme is shown in Figure 1A .
For second-generation spheres, primary spheres were collected by brief centrifugation and incubated with trypsin (0.05%) for 5 min with frequent gentle pipetting to dissociate the spheres. Once the single cells were formed, cells were counted and replated in ultralow attachment plates as described above. Cells were treated with etomoxir 32.5 μM after 24 h of seeding and every 48 h thereafter. The number of spheres was counted after day 6 of treatment.
For second-generation spheres, primary spheres were collected by brief centrifugation and incubated with trypsin (0.05%) for 5 min with frequent gentle pipetting to dissociate the spheres. Once the single cells were formed, cells were counted and replated in ultralow attachment plates as described above. Cells were treated with etomoxir 32.5 μM after 24 h of seeding and every 48 h thereafter. The number of spheres was counted after day 6 of treatment.
4
Spheroid Culture of Prostate Cancer Cells
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Spheroid culture assay was performed as previously described24 (link). In brief, PCa cells were transfected with NC or miR-628 and plated at a density of 2500 cells/well in triplicates in ultralow attachment six-well culture plates (Corning, St. Louis, MO, USA) and grown in stem cell media consisting of DMEM/F12 (Gibco, Cat# 21331020) media supplemented with B27 (Gibco, Cat#17504044) and N-2 (Gibco, Cat# 17502001). Spheres were visualized and counted 10 days after plating using a light microscope. Sphere area was measured using the Zeiss ZEN lite software.
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