Computational protocol: Establishment of xenogeneic serum-free culture methods for handling human dental pulp stem cells using clinically oriented in-vitro and in-vivo conditions

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Protocol publication

[…] To evaluate cell susceptibility to extrinsic stimuli, XFM and SCM cells (5 × 103 cells/cm2) were seeded into 12-multiwell plates and treated with 1 μM staurosporine (Wako Pure Chemical Industries) or 100 μM hydrogen peroxide (H2O2; Wako Pure Chemical Industries) for 6 h at 37 °C, or were exposed to ultraviolet (UV) radiation using a UV transilluminator (UVP, Upland, CA, USA) for 30 min at room temperature. Staurosporine, which is a known chemical inducer of apoptosis, was used as a positive control for cell-damage induction. Cell damage was evaluated by flow cytometry using an Annexin V/propidium iodide (PI) system described previously [], with minor modifications. Briefly, the treated cells were stained with 100 μl of Guava Nexin® reagent (Merck KGaA) for 20 min at room temperature in the dark and subjected to flow cytometry in the Guava™ EasyCyte HT system equipped with Guava™ InCyte software (Merck KGaA). The data were analyzed with FlowJo software (TreeStar). Annexin V-positivity and PI-negativity indicated the fraction of early apoptotic cells and Annexin V/PI-double positivity indicated the fraction of late apoptotic cells. We considered that both fractions represented damaged cells []. The results were expressed as the ratio of the number of damaged cells to the number of nondamaged cells. [...] Statistical analyses were performed using IBM SPSS statistics software (version 23.0; IBM Japan, Tokyo, Japan). All experiments were repeated independently in triplicate, and values were expressed as the mean ± standard deviation (SD). For two-group comparisons, an unpaired t test or a Mann–Whitney U test for nonparametric data was used. The chi-square test was used to compare the presence of BrdU-positive cells. For all statistical analyses, P < 0.01 indicated a statistically significant difference. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Serum Sickness
Chemicals Dimethyl Sulfoxide, Hydrogen Peroxide