For the screening, HT1080 cells were seeded in 40 µl medium in 384-well microplates (CulturPlate, PerkinElmer) with a cell number of 750 cells per well using a MultiFloTM Dispenser (BioTek Instruments). 24 h after seeding, cells were treated with compounds (0.5 µl per well—i.e., 5 µM) or DMSO alone as control. Plate and liquid handling were performed using a HTS platform system composed of a Sciclone G3 Liquid Handler (PerkinElmer) with a Mitsubishi robotic arm (Mitsubishi Electric, RV-3S11) and a CytomatTM Incubator (Thermo Fisher Scientific). The diverse small-molecule library used for this study is composed of 3684 compounds with known mode of action (dissolved in DMSO, 1 mM stock solution). As positive control, cells were treated with the ferroptosis-inhibitor Ferrostatin-1 (2 µM). 4 h later, ferroptosis was induced using 1.5 µM IKE. After 18 h incubation time cell viability was measured by adding 20 µl CellTiter-Glo 2.0 Reagent (Promega). Luminescence signals of the CellTiter-Glo assay were detected on the EnVision 2104 Multilabel plate reader (PerkinElmer). Signals of the Ferrostatin-1-treated wells were set to 100% activity. For hit selection, a threshold of higher than 3 standard deviations from the median of the compound-treated population was set.
Sciclone g3 liquid handler
Manufactured by PerkinElmer
Sourced in United States
The Sciclone G3 Liquid Handler is a precision liquid handling system designed for automated liquid handling tasks in laboratory environments. It features a compact and modular design, enabling customizable configurations to suit various experimental requirements. The Sciclone G3 Liquid Handler is capable of performing accurate and reproducible liquid transfers across a range of volumes, supporting a wide array of labware and sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cytomat incubator, by Thermo Fisher Scientific (5 mentions)
Multiflo dispenser, by Agilent Technologies (5 mentions)
Cellcarrier 384 ultra microplate, by PerkinElmer (2 mentions)
Operetta columbus high content imaging platform, by PerkinElmer (2 mentions)
Rv 3s11, by Agilent Technologies (2 mentions)
Operetta harmony high throughput imaging platform, by PerkinElmer (2 mentions)
Envision 2104 multilabel plate reader, by PerkinElmer (1 mentions)
Culturplate, by PerkinElmer (1 mentions)
Celltiter glo 2.0 reagent, by Promega (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
Felix liquid handler, by Analytik Jena (1 mentions)
Echo liquid handler, by Beckman Coulter (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Zag dna analyzersystem, by Agilent Technologies (1 mentions)
Prosize data analysis software, by Agilent Technologies (1 mentions)
Optiplate, by PerkinElmer (1 mentions)
Flexdrop dispenser, by PerkinElmer (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Cellcarrier plate, by PerkinElmer (1 mentions)
7 protocols using sciclone g3 liquid handler
1
High-Throughput Screening of Ferroptosis Inhibitors
2
PCR Amplification and Capillary Electrophoresis
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PCR amplification of the cloning inserts was done
using Q5 High-Fidelity 2X Master Mix (NEB, Ipswich, MA, catalogue
no. M0492L). Twenty μL reactions were prepared by dispensing
1 μL of each 10 μM reverse primer into the wells of a
96-well PCR plate using the Echo liquid handler (Beckman Coulter,
Brea, CA). A Mastermix consisting of polymerase premix, plasmid DNA
template (pBbB6c, 5–10 ng per reaction), and the single 10
μM forward primer was prepared and dispensed using the FeliX
liquid handler (Analytik Jena, Jena, Germany) or electronic multichannel
pipet. Reactions were run using Touchdown PCR or standard PCR cycling
methods in C1000 thermal cyclers (Bio-Rad, Hercules, CA). Capillary
electrophoresis of PCR products was performed using the ZAG DNA Analyzer
system (Agilent Technologies, Santa Clara, CA). Two μL of each
PCR reaction was electrophoresed using the ZAG 130 dsDNA Kit (75–20 000
bp) or ZAG 110 dsDNA Kit (35–5000 bp) (Agilent Technologies,
catalogue no. ZAG-110–5000; ZAG-130–5000). ZAG sample
plates were prepared using the Sciclone G3 liquid handler (PerkinElmer,
Waltham, MA). ProSize Data Analysis Software (Agilent Technologies)
was used to generate gel images from the sample chromatograms, and
amplicon sizes were estimated by reference to the upper and lower
DNA markers spiked into each sample and a DNA ladder run in well H12
of each sample plate.
using Q5 High-Fidelity 2X Master Mix (NEB, Ipswich, MA, catalogue
no. M0492L). Twenty μL reactions were prepared by dispensing
1 μL of each 10 μM reverse primer into the wells of a
96-well PCR plate using the Echo liquid handler (Beckman Coulter,
Brea, CA). A Mastermix consisting of polymerase premix, plasmid DNA
template (pBbB6c, 5–10 ng per reaction), and the single 10
μM forward primer was prepared and dispensed using the FeliX
liquid handler (Analytik Jena, Jena, Germany) or electronic multichannel
pipet. Reactions were run using Touchdown PCR or standard PCR cycling
methods in C1000 thermal cyclers (Bio-Rad, Hercules, CA). Capillary
electrophoresis of PCR products was performed using the ZAG DNA Analyzer
system (Agilent Technologies, Santa Clara, CA). Two μL of each
PCR reaction was electrophoresed using the ZAG 130 dsDNA Kit (75–20 000
bp) or ZAG 110 dsDNA Kit (35–5000 bp) (Agilent Technologies,
catalogue no. ZAG-110–5000; ZAG-130–5000). ZAG sample
plates were prepared using the Sciclone G3 liquid handler (PerkinElmer,
Waltham, MA). ProSize Data Analysis Software (Agilent Technologies)
was used to generate gel images from the sample chromatograms, and
amplicon sizes were estimated by reference to the upper and lower
DNA markers spiked into each sample and a DNA ladder run in well H12
of each sample plate.
3
High-Throughput Cellular Assay Platform
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Plate and liquid handling was performed using a HTS platform system composed of a Sciclone G3 Liquid Handler from PerkinElmer (Waltham, MA, USA), a MultiFlo™ Dispenser (Biotek Instruments, Bad Friedrichshall, Germany) and a Cytomat™ Incubator (Thermo Fisher Scientific, Waltham, MA, USA) (Schorpp and Hadian, 2014 (link)). Cell seeding and assays were performed in black 384-well CellCarrier-384 Ultra Microplates (PerkinElmer, 6057300). Image acquisition and image-based quantification was performed using an Operetta®/Columbus™ high-content imaging platform (PerkinElmer, USA).
4
High-Throughput Screening of Approved Drugs
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Plate and liquid handling was performed using a HTS platform system composed of a Sciclone G3 Liquid Handler from PerkinElmer (Waltham, MA, USA) with a Mitsubishi robotic arm (Mitsubishi Electric, RV-3S11), a MultiFloTM Dispenser (Biotek Instruments, Bad Friedrichshall, Germany) and a CytomatTM Incubator (Thermo Fisher Scientific, Waltham, MA, USA). Cell seeding and assays were performed in black 384-well CellCarrierTM plates (PerkinElmer, 6007558). The diverse small molecule library used in HTS was acquired from Prestwick Chemical (Illkirch, France). We tested 960 small molecule compounds of the Prestwick Chemical library, which are 100% approved drugs (Food and Drug Administration (FDA), European Medicines Agency (EMA) and other agencies). The purity of the compounds was >90% as reported by the provider of the compounds. Cells were seeded 40h before treatment in 384-well microplates with a cell number of 4 × 104 cells/well. This resulted in about 80–90% confluent after 16 h and in a confluent layer after serum starvation for another 24 h (time of compound addition). Image acquisition and image-based quantification was performed using the Operetta®/Harmony® high-throughput imaging platform (PerkinElmer, USA).
5
High-Throughput Cellular Assay Platform
6
High-Throughput Screening Protocol with AlphaScreen
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We used a HTS platform with an integrated instrumentation for plate and liquid handling. The screening was performed using a Sciclone G3 Liquid Handler from PerkinElmer (Waltham, MA, USA) with a Mitsubishi robotic arm (Mitsubishi Electric, RV‐3S11) and a Flexdrop dispenser (PerkinElmer, Waltham, MA, USA). The AlphaScreen assay was performed in white 384‐well Optiplates™ (PerkinElmer, 6007299). AlphaScreen signal was detected on the EnVision® Multilabel Reader (PerkinElmer, Waltham, MA, USA).
7
High-throughput Cell Imaging Assay
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Plate and liquid handling was performed using an HTS platform system composed of a Sciclone G3 liquid handler from PerkinElmer with a Mitsubishi robotic arm (Mitsubishi Electric, RV-3S11), a MultiFlo dispenser (Biotek Instruments) as well as a Cytomat incubator (Thermo Fisher Scientific). Cell seeding and assays were performed in black 384-well CellCarrier plates (PerkinElmer, 6007558). The plates were coated with gelatin 0.1% for 20 min at 37 °C to facilitate better cell adherence. Cells were seeded in 384-well microplates with 10,000 cells per well. Image acquisition and image-based quantification was done using the Operetta/Harmony high-throughput imaging platform (PerkinElmer). Z′ factors were calculated according to the formula Z′ = 1 − (3(θp + θn)/(µp − µn)), where p is the positive control, n is the negative control, θ is the standard deviation and µ is the mean.
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