Paclitaxel Dose-Response on Brain Organoid Apoptosis

To assess the time-dependent dose-response of paclitaxel exposure on apoptosis induction in mature brain organoids, the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay was used (Promega, Germany). Apoptosis induction is measured by Annexin V binding to trans-localized phosphatidylserine, whereas necrosis is detected by a membrane-impermeable reagent binding to DNA. The sensitivity of the assay was previously tested by the company in 3D cultures (Data available on the company webpage). Furthermore, Annexin-V based assays have been previously used to assess cytotoxicity in 3D cultures (Xiao et al., 2020 (link); Parseh et al., 2022 (link)). Three organoids per batch (day 58 in culture) were incubated with medium, vehicle or paclitaxel (1, 10, 100, 1,000, 10,000 nM) for 14 h, transferred to a white round bottom 96-well ultra-low attachment plate (MS-9096WZ; PHCbi, Netherlands) in NDM and the assay reagents were added according to the manufacturer’s instructions. Luminescence and fluorescence measurements were done 17, 18, 20, 22, 24, 26, 28, 32, 40 and 48 h after the start of the exposure with a SpectraMax iD3 plate reader (molecular devices, San Jose, CA) and normalized to the first measurement (17 h) as well as to the vehicle control. Effects of more than 20% compared to the VC were interpreted as relevant.