Biomek fx

The DIVERSet library, containing 46,000 compounds, was purchased from ChemBridge Corporation (San Diego, CA, USA) and formatted into 384-well mother plates (Beckman Coulter, Brea, CA, USA) using a Biomek FX liquid handler (Beckman Coulter). Assay plate copies (Greiner Bio-One, Kremsmunster, Austria) were subsequently made by dispensing 50 nL of either compound (10 µM final concentration per well) or DMSO from the mother plates into transparent polystyrene 384-well plates using an Echo liquid dispenser (Beckman Coulter). Plates were heat-sealed and stored at −20 °C until use. Dimethyl sulfoxide (DMSO, at matching% v/v) was loaded in columns 1, 2, 23, and 24 as negative controls.

Approximately 5 × 10⁷ PBMCs were thawed and diluted 1:10 in pre-warmed R10 medium and treated with benzonase nuclease HC, purity >99% (Novagen) at 1:10,000. PBMCs were washed twice in PBS before final resuspension in pre-warmed R10 medium. CSM B cells were isolated from PBMCs by depletion of non-target cells using a Switched Memory B cell isolation kit with Pre-Separation Filters and LS columns on a MACS Separator (MACS Miltenyi Biotec). To activate CSM B cells and promote antibody secretion, R10 medium was supplemented with a cocktail of antibodies and cytokines to make complete R10 medium. CSM B cells were resuspended in complete R10 medium at 56 cells/ml and then plated out in 90 μl aliquots (5 cells/well) in ThermoFisher Matrix 384-well plates using a Biomek FX (Beckman Coulter). Cells were incubated at 37 °C, 5% CO₂ for 7 days. On day 7, 30 μl/well of supernatant was replaced with 30 μl fresh complete R10. On day 13, all supernatants were harvested and screened against recombinant N-terminus Hyr1 protein antigen, C. albicans extracted cell walls or C. albicans whole cells from overnight cultures by ELISA. B-cell activation and culturing was monitored by measuring IgG1 concentrations in B-cell supernatants at day 7 and day 13.

Sequence-ready libraries were validated and quantitated on the High Sensitivity D1000 ScreenTape (Agilent Technologies, Santa Clara, CA) to allow for library normalization and equimolar pooling of all study samples on the Biomek FX (Beckman coulter, Brea, CA). Pooled libraries were diluted and loaded at 12 pM on a MiSeq flow cell, with 5% phiX spiked in. Paired end sequencing runs were performed using MiSeq Reagent Kit v2, 500 cycles (Illumina, San Diego, CA).

Compounds were tested on three chordoma cell lines (U‐CH1, U‐CH2 and MUG‐Chor1) using a non‐randomised plate layout in a 96‐well plate format (80 compounds/plate) at a single concentration of 1 µm (n = 3 minimum). Cells were seeded in medium (90 µl/well) using a Multidrop Combi (MDC; Thermo Fisher Scientific, Loughborough, UK) and cultured for 24 h before the compounds were added. The compounds were diluted from 10 mm stocks using an ECHO 550 (Labcyte, CA, USA) to create 10× compound plates and added (10 µl/well) using a Biomek FX (Beckman Coulter, Brea, CA, USA). Cell survival was assessed following 96 h of compound treatment using the water‐soluble tetrazolium salt (WST1) assay (Roche Diagnostics, Burgess Hill, UK) according to the manufacturer's recommendations.

‘PKT assays were performed as described before^{41 (link)}. Briefly, black 96-well µClear plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 10 µg/ml fibronectin (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 1 h at 37 °C. Plates were washed twice with PBS, using a HydroFlex platewasher (Tecan, Männedorf, Switzerland). Subsequently, the plates were coated with white carboxylate modified latex beads (400 nm, 3.25·10⁹ particles per well; Life Technologies, Carlsbad, CA, USA) for 1 h at 37 °C, after which the plate was washed 7 times with PBS. 65 h after siRNA transfection, transfected cells were washed twice with PBS-EDTA and trypsinized. Cells were resuspended into single cell suspensions, then diluted, and finally seeded at low density (~100 cells/well) in the beads-coated plate. Cells were allowed to migrate for 7 h, after which the cells were fixed for 10 min with 4% formaldehyde and washed twice with PBS. For each transfection, duplicate bead plates were generated (technical replicates); transfection of each siRNA library was also performed in duplicate (independent biological replicate). Procedures for transfection, medium refreshment and PKT assay were optimized for laboratory automation by a liquid-handling robot (BioMek FX, Beckman Coulter)’⁴⁰ . (The above text in this paragraph has been similarly described in Fokkelman et al.⁴⁰ ).