96 well u bottom polystyrene plates

Differentiation was performed with the protocol reported before with minor modifications.¹⁴ (link) The procedure is outlined in Figure 2. RxGFP mES cells were dissociated from the flask with 0.25 trypsin and seeded in differentiation medium (OV) (Supplementary Table S1) on 96-well U-bottom polystyrene plates (Greiner Bio-One, Kremsmünster, Austria) in cell density of 3000 cells per well in 50 µL of the media. Cell seeding was performed manually in both experimental groups. Cells were fed with 50 µL of OV supplemented with 1% Matrigel (Corning Life Sciences) manually or automated with WellWash Versa (Thermo Fisher, Waltham, MA, USA) depending on experimental group on day 2 of the differentiation, additional feeding with 50 µL of OV with 0.5% Matrigel was performed on day 5 of differentiation. Further medium change was performed in automated or manual manner with OC media (Supplementary Table S1) starting from day 9 every other day until the day 21.

Two milligrams from the aqueous and ethanolic extracts were fractionated into 48 fractions (1 ml x 48) in a 96 position deep well plate (VWR, Sweden) using a Shimadzu LC-10 system equipped with an SPD-M10AVP Photodiode-Array (PDA) detector. A Phenomenex Jupiter C18 HPLC column (5 μm, 100 Å, 4.6 mm × 250.0 mm) was used for the microfractionation of the crude extracts by employing a gradient of 5–95% CH₃CN (0.05% TFA) over 48 minutes at a flow rate of 1 ml/min. Similarly, 10 mg of the organic extracts were dissolved in 1 ml of 100% CH₃CN and a volume of 200 μl was fractionated into 48 fractions using two consecutive gradients from 5–60% CH₃CN (0.05% TFA) over 20 minutes and 60–95% CH₃CN (0.05% TFA) over 25 minutes at a flow rate of 1 ml/min [20 (link)]. One hundred microlitres from each well was transferred into 96-well U-bottom polystyrene plates (Greiner Bio-one, USA) and dried in a centrifugal evaporator (Savant Speed Vac plus SC110A).