Cellstar 96 well suspension culture plates

The rCHO cells were inoculated at a target viable cell concentration of 0.3 × 10⁶ cells/mL in CELLSTAR 96-well suspension culture plates (Greiner Bio-One, Frickenhausen, Germany) with expansion medium (198 μL). Rows 1 and 12 on the 96-well plates were used as controls with DMSO (2 μL). On day 0, 2 μL of each chemical compound dissolved in DMSO was added to each well. The plates were incubated at 37.0°C with the shaker set to 120 rpm (20-mm stroke length). After 3 days of culture, mAb concentrations were analyzed with Octet QKe (ForteBio, Fremont, CA, USA). The resulting mAb concentrations were converted to arbitrary units according to the following Eq (1).
The compounds for which the arbitrary unit showed a positive value were selected as candidates for the secondary screening. The validity of this screening protocol is shown in S1 Table.

hPOs were seeded in 5 µL BME-2 droplets in sterile CELLSTAR® 96-well suspension culture plates (Greiner Bio-One GmbH) and overlaid with 100 µL hPO medium. hPOs were cultured for 3-5 days prior to experiments to assure organoid formation and growth. For pre-treatment with HOIPIN-8, hPO medium was replaced by hPO medium with the indicated concentrations of HOIPIN-8 and hPOs were placed back in the incubator. After 30 min, hPO medium was again replaced with medium supplemented with the indicated treatments and the plates were transferred to the Zeiss AxioObserver Z1 microscope. Incubation was done at 37 °C and 5% CO₂ during which 24 h time lapse imaging in 30 min intervals was performed. Each well was imaged as tiles of two x two with eleven Z-slices of 60 µm thickness.
For live/dead assays, primary hPOs were stained using 10 µg/mL PI (Millipore Sigma), 0.5 µg/mL FDA (Millipore Sigma) and 200 µg/mL Hoe (ThermoFisher Scientific) for 30 min. Dyes were removed and the wells were washed with pre-warmed PBS prior to imaging in PBS, using the same microscope settings as described above.
Images were exported using the ZEN 2.6 lite software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and subsequently processed using the FiJi/ImageJ software.