P96 1.5h n

Approximately 24 h after seeding, various drug concentrations (500, 100, 50, 10, 1, 0.5, 0.1 and 0 µM) diluted in normal culture media were transferred to G18-T or FWP1 cells plated in 384-well plates by utilizing the OT-2 liquid handler robot and associated software (Opentrons, Brooklin, NY, USA). Custom labware templates for 96-well plate (cat#P96- 1.5H-N, Cellvis, Mountain View, CA, USA), 384-well plate and reservoir were created using the measurements provided by the manufacturer for each item using the Opentrons labware creator. Moreover, a protocol script was created in Python that enable transfer of the drugs to tumor cells cultured in 384-well plates, incorporating the GEN1 single channel p300 pipette and a temperature module to maintain cells at 37 °C, while drugs were transferred to each plate. Before running the protocol, 0.5 mM (500 µM) of 67 drugs were individually diluted in StemPro NSC medium in the wells of row A of seven 96-well plates from the 10 or 2 mM stock concentration of each drugs. Then, the protocol was run through the steps as detailed in Figure S2, and, after drug addition, multiwell plates were incubated in a humidified environment at 37 °C and 5% CO₂ for 72 h before viability measurements.

These procedures followed the commonly used cell labeling method^{19 (link),53 (link)}, similar to our previous work^{25 (link)}. The HeLa cells were cultured in 96-well glass-bottom plates (thickness 170 ± 5 μm, P96-1.5H-N, Cellvis) with 15,000 cells in each unit, with the temperature set at 37 °C, the humidity at 95%, and the CO₂ concentration at 5% overnight (>12 h). The cells were rinsed three times using 200-μL pre-warmed PBS buffer (37 °C, 5 min each) to remove the residual culture media and then fixed using PFA for 15 min at room temperature. Subsequently, the cells were rinsed three times using PBS buffer to remove the residual PFA. Next, the cells were permeabilized in 200-μL 0.2% Triton X-100 for 30 min at room temperature and rinsed three times using HEPES buffer. 200 μL Quick Block blocking buffer was added to block the fixed cells for 20 min at room temperature. When the procedure of blocking was completed, 150 μL of freshly prepared phalloidin-bioconjugated lanthanide-doped nanoparticles (with 1% bovine serum albumin in buffer (200 μg mL⁻¹) were added. The cells were kept at room temperature for 4 h and then rinsed twice with pre-warmed (37 °C) HEPES buffer. Finally, the HEPES buffer was removed and a small drop of embedding medium was dropped into the well. The sample was kept at room temperature and in the dark for 24 h to ensure complete dryness before measurement.