96 well ultra low attachment microplate

After treatment with doses of 0-10 Gy IR, 500 single cells/well of ML cultures were seeded on 6-well microplates (Corning), three wells per condition; on day 14, the colonies formed were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. For SP cultures, 100 single cells/well were seeded on 96-well ultra-low attachment microplates (Corning), 12 wells per condition; on day 7, only the spheres formed were counted. Colonies or spheres larger than 70 μm were qualified as deriving from a single cloning cell. Plating efficiency (PE) and survival fraction (SF) after the dose were calculated using equations (1) and (2). The clonogenic assay for ML cultures was read on day 14 to render the growth of colonies more evident; the SP cultures were read at day 7 because, after this time, indistinguishable agglomerates are formed. The median lethal dose (LD50) was obtained employing an exponential mathematical model.
$\begin{array}{c}\text{PE}=\frac{\text{Number\hspace{0.17em}of\hspace{0.17em}colonies\hspace{0.17em}or\hspace{0.17em}spheres\hspace{0.17em}by\hspace{0.17em}control}}{\text{Number\hspace{0.17em}of\hspace{0.17em}seeded\hspace{0.17em}cells}},\end{array}$
$\begin{array}{c}\text{SF}=\frac{\text{Number\hspace{0.17em}of\hspace{0.17em}colonies\hspace{0.17em}or\hspace{0.17em}spheres\hspace{0.17em}formed\hspace{0.17em}after\hspace{0.17em}treatment}}{\left(\text{Number\hspace{0.17em}of\hspace{0.17em}seeded\hspace{0.17em}cells}\right)\left(\text{PE}\right)}.\end{array}$

Cells were seeded in 96-well ultra-low attachment microplates (Corning) at 500 cells per well and grown for 72 h. After 72 h the indicated concentration of drug was added, and the spheroids were grown for an additional 72 h. At the end of the 6-day time course spheroids were co-stained with NucBlue Live ReadyProbes reagent (Thermo Fisher, 37605) and 2.0 μM Calcein AM Green for two hours and then imaged by confocal microscopy on a Cellinsight CX7 high content analysis platform. For each spheroid, 15 confocal 20-micron Z-stacks were imaged in both the blue and green channels to visualize NucBlue-stained nuclei and Calcein AM Green-stained cytosol, respectively. The resulting 30 images were then assembled into a single image projection and analyzed using HCS Studio cell analysis software (Thermo Fisher). Total spheroid visible area was calculated by delineating the boundary of the spheroid in the green channel.

The stroma-rich 3D tumor spheroids were prepared as previously described 38 (link). Briefly, a mixture of PANC-1 tumor cells and NIH 3T3 fibroblast cells at a ratio of 2:1 was suspended in Spheroid Formation ECM (Amsbio, Cambridge, MA) according to the manufacturer's instructions and then added to the Corning 96-well ultralow attachment microplates (Pittsburgh, PA). The cells were incubated at 37ºC to form stroma-rich tumor spheroids. After 7 days of incubation, the stroma-rich tumor spheroids were incubated with Cy5-siRNA nanocomplexes at 37ºC for 2 h, followed by washes with DPBS and fixation with 4% paraformaldehyde. The tumor spheroids were then visualized using a confocal microscope to evaluate the penetration of the siRNA nanocomplexes.

To form spheroids, 5 × 10³ HOS cells and 2 × 10³ 143B cells were seeded per well into a 96-well ultra-low attachment microplate (Corning; #7007), centrifuged at 125 × g for 10 min at RT, and cultured for 3 days. After spheroid formation, plates were chilled on ice for 10 min before the addition of 100 μL of Matrigel (Corning; #354234) to a final concentration of 4 mg/mL in each well. The plate was incubated at 37 °C for 30 min to allow Matrigel to polymerize and 50 μL of culture medium was then added to each well. Images were taken after addition of Matrigel (Day 0) and 3–4 days later (Day 3 for 143B cells or Day 4 for HOS cells) using a Leica DMIRB inverted microscope and N Plan 5x/0.12 PH0 objective. Spheroid area was measured manually using ImageJ, and the final area (Day 3 or 4) was divided by the initial area (Day 0) to obtain a fold-change for each spheroid.

To prepare tumor spheroids, SJSA-1 and 143B cells (5 × 10³ cells/well) were cultured in a Corning® 96-Well Ultra Low Attachment Microplate. After 5–7 days, uniform and compacted spheroids were selected for the following studies. The spheroid-derived cells were used as stem-like osteosarcoma cells. The monolayer cultured cells were used as non-stem-like osteosarcoma cells. For the uptake study, the tumor spheroids were incubated with free coumarin-6 or NP_{coumarin 6} for 30 min, and then the spheroids were rinsed with cold PBS and fixed with 4% paraformaldehyde for 15 min. Fluorescence intensity was observed under a fluorescent microscope and further quantified with flow cytometry (BD LSRFortessa). For the tumor spheroid formation assay, DMSO, J4, Apa, and J4+Apa were added to each well. The morphology of the spheroids was observed under a microscope. Cell apoptosis was determined using flow cytometry.

A single cell was cultured in a 96-well ultra-low attachment microplate (Corning) for 15 days, and the status of cells was recorded by light microscope. At various time points after culture, the percentage of tumorsphere formation was calculated.