Journal: Scientific reports

Article Title: Stem cell enriched-epithelial spheroid cultures for rapidly assaying small intestinal radioprotectors and radiosensitizers in vitro.

doi: 10.1038/s41598-018-33747-7

Figure Lengend Snippet: Figure 1. Spheroid formation assay to identify GI radioprotectors. Schematic representation illustrating how stem cell-enriched epithelial spheroid cultures are established (panel a). Schematic outlining how the spheroid formation assay (SFA) is performed (see text for details, panel b). Z-stack images of treated stem cell-enriched epithelial spheroid cultures are stitched together to visualize spheroids (panel c). A representative image of the stitched z-stack is shown (panel d). Stem cell-enriched epithelial spheroid cultures treated with vehicle (PBS) or 2 mM WR-1065 for 2 h were exposed to the indicated doses of ionizing radiation. Spheroids were immediately dissociated into single cells, replated in Matrigel and imaged 5 days later (panel e) (Scale bars, 1000 μm). Spheroids larger than 150 μm in diameter were quantified and mean surviving fraction is plotted. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed, Student’s t test (N = 3 per group). Error bars are ± SEM.

Article Snippet: The following equipment and consumables were used to develop the SFA: Cellometer® Vision Cell Profiler CBA (Automated Cell Counter); CLARIOstar (BMG Labtech); Cytation 3 (Biotek); Clear cell culture plate, 24 well (Thermo-Fisher); Black wall clear bottom cell culture assay plate, 24 well (MIDSCI); 5 ml Falcon® round bottom tubes with cell strainer cap (Corning); Cellometer® Disposable Counting Chambers (Nexcelcom); X-RAD 320 Biological Irradiator (Precision X-Ray), VX-2500 Multi-Tube Vortexer (VWR).

Techniques: Tube Formation Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Stem cell enriched-epithelial spheroid cultures for rapidly assaying small intestinal radioprotectors and radiosensitizers in vitro.

doi: 10.1038/s41598-018-33747-7

Figure Lengend Snippet: Figure 3. Spheroid Formation Assay for identifying radioprotectors in vitro. Stem cell-enriched epithelial spheroid cultures were incubated in the presence of vehicle or 2 mM WR-1065 for 2 h and then either mock irradiated or exposed to increasing doses of ionizing radiation. Spheroids were immediately dissociated to single cells and 3,000 cells were plated per well in triplicate and cultured for 5 days. Representative bright field images (panel a) (Scale bars, 1000 μm) and corresponding bioluminescence measurements (panel b) are shown for day 5 samples. **P < 0.01, ***P < 0.001 by two-tailed, Student’s t test (N = 3 per group). Error bars are ± SEM.

Article Snippet: The following equipment and consumables were used to develop the SFA: Cellometer® Vision Cell Profiler CBA (Automated Cell Counter); CLARIOstar (BMG Labtech); Cytation 3 (Biotek); Clear cell culture plate, 24 well (Thermo-Fisher); Black wall clear bottom cell culture assay plate, 24 well (MIDSCI); 5 ml Falcon® round bottom tubes with cell strainer cap (Corning); Cellometer® Disposable Counting Chambers (Nexcelcom); X-RAD 320 Biological Irradiator (Precision X-Ray), VX-2500 Multi-Tube Vortexer (VWR).

Techniques: Tube Formation Assay, In Vitro, Incubation, Irradiation, Cell Culture, Two Tailed Test

Journal: Scientific reports

Article Title: Stem cell enriched-epithelial spheroid cultures for rapidly assaying small intestinal radioprotectors and radiosensitizers in vitro.

doi: 10.1038/s41598-018-33747-7

Figure Lengend Snippet: Figure 4. Spheroid Formation Assay for identifying radiosensitizers in vitro. Stem cell-enriched epithelial spheroid cultures were incubated in the presence of vehicle (DMSO) or 40 nM SN-38 for 24 h and then either mock irradiated or exposed to increasing doses of ionizing radiation. Spheroids were immediately dissociated to single cells and 3,000 cells were plated per well in triplicate and cultured for 5 days. Representative bright field images (panel a) (Scale bars, 1000 μm) and corresponding bioluminescence measurements (panel b) are shown for day 8 samples. *P < 0.05 by two-tailed, Student’s t test (N = 3 per group). Error bars are ± SEM. Stem cell-enriched epithelial spheroid cultures were incubated in the presence of DMSO or 40 nM SN-38 for 24 h and then either mock irradiated or exposed to 2 Gy IR. Spheroids were harvested 3 h later and stained for γH2AX or cleaved caspase-3. Representative images are shown. Scale bars, 100 uM and 10 uM (inset) (Panel c).

Article Snippet: The following equipment and consumables were used to develop the SFA: Cellometer® Vision Cell Profiler CBA (Automated Cell Counter); CLARIOstar (BMG Labtech); Cytation 3 (Biotek); Clear cell culture plate, 24 well (Thermo-Fisher); Black wall clear bottom cell culture assay plate, 24 well (MIDSCI); 5 ml Falcon® round bottom tubes with cell strainer cap (Corning); Cellometer® Disposable Counting Chambers (Nexcelcom); X-RAD 320 Biological Irradiator (Precision X-Ray), VX-2500 Multi-Tube Vortexer (VWR).

Techniques: Tube Formation Assay, In Vitro, Incubation, Irradiation, Cell Culture, Two Tailed Test, Staining