Ultra low attachment 96 well plates

To determine the sensitization potential of compounds, HOC/ADR and MCF-7/PAX were seeded into the ultra-low attachment 96-well plates (Sigma-Aldrich) at a cell density 0.5 × 10⁵ cells/mL. After 3 days of spheroid formation, spheroids were carefully washed once with PBS and fresh DMEM medium (99 µL) containing the appropriate concentration of compounds K3, K4, K7 was added. Using a binary serial compound dilution, the concentration range of both cytostatics was prepared in the new 96-well plates (0.06–8 mM). After 1 µL transfer of cytostatics into the corresponding cell line, the final concentration range were 0.6–80 µM. The required volume of DMSO (1% V/V) was added to the positive control. After an incubation of 72 h, 100 µL of CellTiter-Glo 3D Reagent (Promega) was added to each well. Plates with spheroids were incubated for 25 min at room temperature. Afterwards, the luminescent signal was recorded.

U87 (7×10³) cells were seeded in ultra-low attachment 96-well plates (Sigma-Aldrich) and left for 2 days to form spheroids (diameter 250 μm). Spheroids were treated with CMPD1 for 72 h and cell viability was determined by AlamarBlue assay (as described above). Alternatively, spheroids were dispersed using Accumax (Millipore), cells (4×10³) were seeded into 10 cm Petri dishes and allowed to form colonies for 12–15 days. Colonies (50 cells=1 colony) were counted using ImageJ sofware (NIH).

Mouse E14 ESCs (male) (Hooper et al., 1987 (link)) were cultured in naïve or primed conditions as described below. Dnmt3a/b knock-out ESCs (male) (von Meyenn et al., 2016 (link)), Tet1-3 knock-out ESCs (male) (Hu et al., 2014 (link)) and Tdg knock-out ESCs (male) (Kunz et al., 2009 (link)) were grown in primed conditions as described below.
Naïve ESCs were cultured in serum free media with 2i inhibitors (Neurobasal, N2, B27, 103 U/ml LIF, 1 μM Mek inhibitor PD0325901 and 3 μM Gsk-3β inhibitor CHIR99021) without feeders at 37°C and 5% CO₂.
Primed ESCs were cultured in serum containing media (DMEM 4,500 mg/l glucose, 4 mM L-glutamine, 110 mg/l sodium pyruvate, 15% fetal bovine serum, 1 U/ml penicillin, 1 μg/ml streptomycin, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, and 103 U/ml LIF ESGRO) without feeders at 37°C and 5% CO₂.
For the 2i release experiment, ESCs in naïve culture conditions were washed with PBS before addition of media for primed ESC growth.
EB differentiation was performed by seeding 1000 primed ESCs per well in ultra-low attachment 96-well plates (Sigma-Aldrich) in primed ESC culture media without Lif.