Freedom evo 200

The esiRNA screening was performed on Tecan Freedom EVO 200 Automated workstation (Tecan Group Ltd., Switzerland, RRID:SCR_016771), equipped with RoMa (Robotic Manipulator Arm) and LiHa (Liquid Handling Arm) with 1 ml dilutors. Briefly, ESCs were reversely transfected with esiRNAs and Lipofectamine RNAiMAX (Invitrogen 56532) in 96-well plates. Subsequently, cells were treated with APH or ATRi 36 hr post-transfection. Twelve hours after treatment, cell proliferation was assessed by CyQUANT Cell Proliferation Assay kit (ThermoFisher C35011), followed by cell lysis using Cells-to-CT Kit (ThermoFisher, AM1728). The Taqman qPCR assays were performed using Biomek FXP Automated Workstation (Beckman Coulter Life Sciences).

FP reactions were conducted in black 384 well plates using the TECAN Freedom EVO 200 dispenser (Tecan). We dispensed 3x working concentrations of BRCA1 tBRCT protein and TAMRA-labeled BACH1 peptide, 10μl each prepared in assay buffer (20mM Tris buffer pH 7.4, 200mM NaCl, 0.05% Tween-20, 2mM DTT) to achieve final concentrations of 75nM and 10nM, respectively. 10 μl of compound at a final concentration of 125μM was added to the plate and incubated for 20 min at room temperature. Relative fluorescence was measured using the TECAN infinite M1000 Pro microplate reader using an excitation wavelength 530 nm, and an emission wavelength of 610 nm. The degree of polarization was expressed in millipolarization units (mP) as calculated by the reader software using fluorescence intensities parallel and perpendicular with the plane of linearly polarized excitation light. 1% DMSO controls were used. Test compounds were assayed in triplicate. Percent inhibition was calculated to express compound activity after normalising to controls.

Supernatants from HuALN^® reactors were collected over a period of 28 days. Since (re-) stimulation took place after the collection of supernatants, changes in cytokine secretion can be detected one day later, at the earliest. Supernatants were analyzed with a multiplexed suspension array system (Bio-Plex 200, Bio-Rad, Munich, Germany). A Bio-Plex Express assay (Bio-Rad) for six custom analytes was used to quantify the cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ, and TNF-α. The assay was performed according to the manufacturer’s protocol, but conducted fully-automated on a Tecan Freedom Evo 200 (Tecan, Männedorf, Switzerland) with an in-house-developed Tecan Freedom EVOware^® script in order to lower the variance from manual handling [27 (link)]. All samples were tested in duplicate. The results were analyzed with Bio-Plex Manager 6.1 (Bio-Rad, Munich, Germany) using the logistic five-point regression method.

The holdup assay was performed on a Tecan freedom Evo200 robot with 384-well plates in singlicate for the three 11-mer PTEN variants and the 13-mer PTEN peptide as described in [31 (link),32 (link)]. Briefly, prior to interaction assay, 2.5 μL of streptavidin resin was saturated in each well with 20 μL of biotinylated PBM peptides (42 μM) and then washed twice with an excess of free biotin, while the reference resin was incubated only with biotin. Right before the holdup experiment, the PDZ library was spiked with an internal standard of lysozyme. Then, the biotin- or PBM-saturated resin was incubated, each in a distinct well of a 384-well plate, with complete cell lysates diluted so that the concentration of the tag-PDZ present in the crude extract is adjusted at 4 μM. After a sufficient time to reach complex equilibrium (15 min.), a fast and mild filtration step is performed and the tag-PDZ concentrations were measured by capillary electrophoresis instrument (LabChip GXII, PerkinElmer, Massachusets, USA). Standard markers were used to convert migration time into molecular weight on the LabChip software and inappropriate molecular weight markers were corrected or excluded.

Hep G2 and LLC-PK1 cells were seeded in 96 well plates (Nunc, 3598) with 5 × 10⁴ and 2.5 × 10⁴ cells/well, respectively. After seeding, the cells were incubated overnight (37 °C, 85% relative humidity, 5% CO₂) to allow cell adhesion. Sample dilution and addition were performed using a Tecan Freedom EVO 200 robotic system. The cells were incubated for 48 h with the different NPs (0.046–300 (600 for 1-pentyl cyanoacrylate (1-PCA), 2-heptyl cyanoacrylate (2-HPCA) and 3,3-dimethylbutyl cyanoacrylate (3,3-DMBCA)) μg/mL). The MTT assay was performed using a fully automated method on a Beckman FX^P robotic system, integrated with CO₂ incubator, plate reader and a SCARA arm. In short, cell medium was aspirated and exchanged with 250 μL of medium containing a final concentration of 1 mg MTT/mL. The incubation was continued for 3 h at 37 °C to allow formation of the formazan-particles, after which medium was discarded and formazan particles were dissolved in DMSO with 0.1 M glycine and 0.1 M NaCl. The absorbance was read by a plate reader (SpectraMax I3X, Molecular Devices) at 570 nm, and background from absorbance at 650 nm was subtracted.