The SiGENOME SMARTpool siRNA library (targeting the 18,107 genes of the whole human genome) was screened in HEK293 cells stably over-expressing a mCherry-APP695wt-YFP. Briefly, 50 nl of 10 µM solutions of each siRNA were first transferred to 384-well microtiter plates using an Echo 555 liquid handler. Next, 10 µl of D-PBS containing 0.1 µl of lipofectamine™ RNAiMax were then distributed using a BioTEK EL406 Washer Dispenser. After a 30-min incubation at room temperature (enabling the transfectant complex reaction), 40 µl of HEK293-mCherry-APP695wt-YFP cells were distributed onto the plates using a BioTEK EL406 Washer Dispenser, in order to obtain a final density of 3000 cells per well. The microplates were incubated for 3 days at 37 °C. The cells were then incubated with 5 µg/ml of Hoechst 33,342 at 37 °C with 5% CO2 (v/v) for 30 min. After removal of the cell medium, 10% formalin was added to each well and plates were incubated at room temperature for 30 min for staining and cell fixation. Lastly, the cells were stored in D-PBS, and images were acquired at 405, 488 and 561 nm with an InCell Analyser 6000 high-resolution automated confocal microscope. One field per well was read from the B1 well to the O24 well in a horizontal, serpentine acquisition mode with a 20× objective.
El406 washer dispenser
Manufactured by Agilent Technologies
Sourced in Germany, United States
The EL406 Washer Dispenser is a multi-functional laboratory equipment designed for automated liquid handling tasks. It features a high-precision dispensing system and flexible configuration options to accommodate a variety of microplate formats and liquid volumes.
Automatically generated - may contain errors
Lab products found in correlation
Analyser 6000, by INCELL (2 mentions)
Synergy h1 hybrid, by Agilent Technologies (2 mentions)
Goat anti higg fc hrp, by Merck Group (2 mentions)
Precision xs microplate sample processor, by Agilent Technologies (2 mentions)
Biostack microplate stacker, by Agilent Technologies (2 mentions)
Synergy h2 microplate reader, by Agilent Technologies (2 mentions)
Acumen explorer, by SPT Labtech (2 mentions)
Incell2000 hca platform, by GE Healthcare (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
384 well assay plate, by Corning (1 mentions)
Lullaby stem, by Ozyme (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Tetramisole hydrochloride, by Merck Group (1 mentions)
Sealplate, by Merck Group (1 mentions)
Echo 550, by Beckman Coulter (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
Celltiter glo, by Promega (1 mentions)
M1000, by Tecan (1 mentions)
Black 96 well imaging plates, by Greiner (1 mentions)
19 protocols using el406 washer dispenser
1
Screening of siRNA Library in HEK293 Cells
2
Screening siRNA Library in APP-Expressing Cells
3
High-Throughput Screening of miRNA Modulators in hiPSC-CMs
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A high-throughput screening (HTS) by using a miRNA-library (Thermo Fisher Scientific/Ambion; mirVana Human Library v19.0; 2019 miR-mimics and miR-inhibitors) was performed in two individual screenings. Three thousand and five hundred hiPSC-CM/well were plated on Geltrex (Thermo Fisher Scientific) -coated 384-well plates (Corning) using an EL406 washer dispenser (BioTeK) in a laminar flow work cabinet to avoid contamination. For each miRNA, 400 nM dilution of the miRNA were complexed with Lullaby Stem (OZ Biosciences) according to the manufacturer protocol. The mixture was transferred to the 384-well assay plate (Corning) using a Freedom EVO liquid handling workstation (Tecan) resulting in a final concentration of 60 nM miRNA in a total volume of 50 μL per well with a slow dispense speed (1 μL/s). miRNA transfected hiPSC-CM were kept for 24 h at 37°C in 5% CO2. Medium exchange was performed by an EL406 washer dispenser (BioTeK) after 24 h 48 h post transfection, hiPSC-CM were exposed to transient hypoxia (0.5% oxygen, 5% CO2) for 2 h. Thereafter, cells were replaced with cardio culture medium containing EdU and incubated for 48 h. Thereafter, cells were fixed and processed for immunofluorescence analysis.
4
Multiplex biomarker assessment in study
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Plasma levels of MPO, sCD14, sCD163, granzyme B, CX3CL1 (fractalkine) and TIM-3 (T cell immunoglobulin and mucin domain 3) and serum levels of sCD25 were measured by enzyme linked immunosorbent assay (ELISA) in duplicate using commercially available antibodies (R&D Systems, Minneapolis, MN, USA) in a 384 format using the combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT, USA) dispenser/washer (EL406). Absorption was read at 450 nm with wavelength correction set to 540 nm using an ELISA plate reader (Synergy H1 Hybrid, Biotek, Vinooski, VT, USA). The intra- and interassay coefficient of variation were < 10% for all assays.
5
Plasma Leptin Measurement by EIA
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University Hospital, Rikshospitalet. Plasma samples were thawed in a water bath at 37°C for 5 minutes, followed by centrifugation for 2 minutes at 13500g to obtain platelet-free plasma.
Plasma levels of leptin were measured in duplicates by enzyme-immunoassay (EIA) using commercially available reagents (R&D Systems, Minneapolis, MN) in a 384 format using the combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT) dispenser/washer (EL406). Absorption was read at 450 nm with a wavelength correction set to 540 nm using an EIA plate reader (Synergy H1 Hybrid, BioTek, Vinooski, VT). The intraand inter-assay coefficients of variation were 1.7% and 6.4%, respectively.
Plasma levels of leptin were measured in duplicates by enzyme-immunoassay (EIA) using commercially available reagents (R&D Systems, Minneapolis, MN) in a 384 format using the combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT) dispenser/washer (EL406). Absorption was read at 450 nm with a wavelength correction set to 540 nm using an EIA plate reader (Synergy H1 Hybrid, BioTek, Vinooski, VT). The intraand inter-assay coefficients of variation were 1.7% and 6.4%, respectively.
6
Automated High-Throughput Microscopy of Worms
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In order to have clear images, we washed the plates by sequential flushes of water, shaking to disaggregate the bacteria, sedimentation of the worms and aspiration of the supernatant (EL406 washer dispenser, Biotek). Prior to this, we added 10 μl of tetramisole hydrochloride (100 mM, Sigma-Aldrich) to each well to paralyse the worms. Each well was filled to the brim and sealed with transparent SealPlate (Sigma-Aldrich) to ensure the horizontal meniscus required to give uniform brightfield illumination across each well. We acquired pictures in brightfield, green and/or red channel with the IN Cell Analyzer 2000 (GE Healthcare) using a 2× objective in order to have the entire well in one image. A whole 96-well plate can be imaged in two channels in less than 10 min and in three channels in less than 15 min.
7
SARS-CoV-2 Antibody Titration ELISA
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For titration ELISA, purified IgGs were titrated from 3.18 μg/mL- 0.001 ng/mL on 30 ng/well of the following antigens: S1-S2-His (High Five cell produced), RBD-mFc (High Five cell produced), S1-mFc (High Five cell produced) and TUN219-2C1-mFc (as control for unspecific Fc binding). In addition, all scFv-hFc were also tested only at the highest concentration (3.18 μg/mL) for unspecific cross-reactivity on Expi293F cell lysate (104 cells/well), BSA (1% w/v) and lysozyme. IgGs were detected using goat-anti-hIgG(Fc)-HRP (1:70000, A0170, Sigma). Titration assays were performed using 384 well or 96 well microtiter plates (Greiner Bio-One) using Precision XS microplate sample processor (BioTek), EL406 washer dispenser (BioTek) and BioStack Microplate stacker (BioTek). EC50 were calculated with by GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve. Titration ELISAs on other coronaviruses and S1-HIS mutants were performed as described above.
8
Multiplex Serum Biomarker Profiling
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Peripheral venous blood was drawn into pyrogen‐free tubes without any additives. After coagulation at room temperature, tubes were centrifuged at 1500 g for 10 min, and serum was stored at –80°C in multiple aliquots. Serum levels of CRP (Cat. no. DY1707), PTX3 (Cat. no. DY1826), sTNFR1 (Cat. no. DY225), OPG (Cat. no. DY805), DLL1 (Cat. no. DY1818), CXCL16 (Cat. no. DY1164), Axl (Cat. no. DY154), ePCR (Cat. no. DY2245), endostatin (Cat. no. DY1098), GDF‐15 (Cat. no. DY957), CatS (Cat. no. DY1183), CD147 (Cat. no. DY972), CCL18 (Cat. no. DY394), Gal3BP (Cat. no. DY2226), sCD163 (Cat. no. DY1607) and sCD166 (Cat. no. DY656) were measured in duplicate by enzyme‐linked immunosorbent assay with antibodies obtained from R&D Systems (Minneapolis, MN, USA) in a 384 format using a combination of a CyBi SELMA (CyBio, Jena, Germany), EL406 washer/dispenser (Biotek, Winooski, VT, USA) and Synergy H2 microplate reader (Biotek). vWF was measured by the same method with antibodies obtained from DakoCytomation (Glostrup, Denmark). The intra‐ and interindividual coefficients of variation were <10%.
9
Fluorometric Microculture Cytotoxicity Assay Protocol
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Concomitant and anticancer drugs were added to the cell plates by an Echo 550 (Beckman Coulter, California, USA) after the cells had been seeded and preincubated overnight. Anticancer drugs were added approximately 4 h after the addition of concomitant drugs. After drug addition, the cells were incubated for 72 h before assessment of cell viability in the fluorometric microculture cytotoxicity assay (FMCA).
The FMCA is based on the hydrolysis of fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes [10 (link)]. After washing the plates with PBS twice, FDA was added into each well by an EL406 washer dispenser (BioTek Instruments Inc, Vermont, USA). The cells were then incubated for 50 min to hydrolyze FDA. The fluorescein signals were then measured by the CLARIOstar microplate reader (BMG Labtech, Germany) [10 (link)].
The FMCA is based on the hydrolysis of fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes [10 (link)]. After washing the plates with PBS twice, FDA was added into each well by an EL406 washer dispenser (BioTek Instruments Inc, Vermont, USA). The cells were then incubated for 50 min to hydrolyze FDA. The fluorescein signals were then measured by the CLARIOstar microplate reader (BMG Labtech, Germany) [10 (link)].
10
High-Throughput Screen of FDA-Approved Drugs
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A549 cells were trypsinized, resuspended in RPMI or ftABS, then 500 cells were plated in duplicate in 50 μL of medium per well of 384-well plates (Corning 3570BC) using an EL406 Washer Dispenser (BioTek). Cells were allowed to attach overnight, after which 50 nL of each compound from the SCREEN-WELL® FDA approved drug library V2 (Enzo Life Sciences, BML-2843) was dispensed into each well using a Freedom Evo 150 Liquid Handler (Tecan) mounted with a 384W pin tool (V&P Scientific) at doses ranging from 10 μM to 1 nM via 5-pt dose titration. Cells were treated for 72 hr, after which 10 μL of CellTiter-Glo (Promega, G7572) reagent was added to each well, incubated at room temperature for 10 min, and luminescence read using Tecan M1000 plate reader. Luminescence values were normalized to DMSO-treated wells of the appropriate medium, and the area under the curve was quantified to assess the activity of each compound.
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