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.
Multiflo dispenser
Manufactured by Agilent Technologies
Sourced in United States
The MultiFlo dispenser is a liquid handling instrument designed for precise and automated dispensing of small volumes of liquids. It features multiple independent dispenser channels for simultaneously dispensing different reagents or samples into microplates or other labware. The MultiFlo dispenser is capable of accurately and consistently delivering volumes ranging from microliters to milliliters, making it a versatile tool for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytomat incubator, by Thermo Fisher Scientific (5 mentions)
Sciclone g3 liquid handler, by PerkinElmer (5 mentions)
Rv 3s11, by Agilent Technologies (2 mentions)
Operetta harmony high throughput imaging platform, by PerkinElmer (2 mentions)
Cellcarrier 384 ultra microplate, by PerkinElmer (2 mentions)
Operetta columbus high content 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)
Pen strep glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Recombinant luciferase, by Promega (1 mentions)
Bright glo, by Promega (1 mentions)
Envision microplate reader, by PerkinElmer (1 mentions)
Operetta, by PerkinElmer (1 mentions)
Echo 550 acoustic dispenser, by Beckman Coulter (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
D300e digital dispenser, by Tecan (1 mentions)
Ruxolitinib, by Cayman Chemical (1 mentions)
Stattic, by Cayman Chemical (1 mentions)
15 protocols using multiflo dispenser
1
High-Throughput Screening of Ferroptosis Inhibitors
2
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).
3
Quantifying Luciferase Inhibition Screening
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To quantify the rate
of false positives reported during the PbLuc liver stage reconfirmation
screen, hits selected for tertiary round screening (405 compounds
from fresh DMSO stocks) were also tested for luminescent interference
between recombinant firefly luciferase and luciferin. Initially, 40
nL of selected compounds was dispensed using a Gen 4 Plus Acoustic
Transfer System (Biosero, San Diego, USA) at 1:3 in 12-point dose
response format (25 to 141.13 × 10–6 μM;
final DMSO concentration of 0.5% per well) in 1536-well, white, opaque-bottom
plates (ref# 789173-F, Greiner Bio-One). A 24-point single concentration
series of Luciferase Inhibitor-II (9.8 μM per well) was dispensed
as a positive control, while 96 wells of DMSO at 0.5% per well acted
as the negative control. Separately, a solution of 20 pM recombinant
luciferase (Promega cat# E170A) was prepared on ice by successive
dilutions (first 1:999 then into final solution at 20 pM) in phenol
naive DMEM (Invitrogen cat# 31053-028), 5% FBS (Corning), and 5×
Pen Strep Glutamine (Invitrogen). This solution was dispensed at 8
μL per well into the previously spotted plates using a MultiFlo
dispenser (BioTek) and incubated at room temperature for 1 h prior
to the addition of 1 μL of BrightGlo (Promega). Immediately
after the addition of BrightGlo, plates were read using the EnVision
Microplate reader (PerkinElmer).
of false positives reported during the PbLuc liver stage reconfirmation
screen, hits selected for tertiary round screening (405 compounds
from fresh DMSO stocks) were also tested for luminescent interference
between recombinant firefly luciferase and luciferin. Initially, 40
nL of selected compounds was dispensed using a Gen 4 Plus Acoustic
Transfer System (Biosero, San Diego, USA) at 1:3 in 12-point dose
response format (25 to 141.13 × 10–6 μM;
final DMSO concentration of 0.5% per well) in 1536-well, white, opaque-bottom
plates (ref# 789173-F, Greiner Bio-One). A 24-point single concentration
series of Luciferase Inhibitor-II (9.8 μM per well) was dispensed
as a positive control, while 96 wells of DMSO at 0.5% per well acted
as the negative control. Separately, a solution of 20 pM recombinant
luciferase (Promega cat# E170A) was prepared on ice by successive
dilutions (first 1:999 then into final solution at 20 pM) in phenol
naive DMEM (Invitrogen cat# 31053-028), 5% FBS (Corning), and 5×
Pen Strep Glutamine (Invitrogen). This solution was dispensed at 8
μL per well into the previously spotted plates using a MultiFlo
dispenser (BioTek) and incubated at room temperature for 1 h prior
to the addition of 1 μL of BrightGlo (Promega). Immediately
after the addition of BrightGlo, plates were read using the EnVision
Microplate reader (PerkinElmer).
4
High-Content Imaging of Malaria Gametocytes
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Prior to parasite addition,
10 nL (final concentration of 2 μM) of each compound was dispensed
using an Echo 550 acoustic dispenser (Labcyte) in 1536-well, black,
clear-bottom plates (ref# 789866-691-2B, Greiner Bio-One). High-content
imaging experiments were performed on stage V gametocytes as previously
described.51 (link) Briefly, mature gametocytes
were diluted to 0.5–0.75% gametocytemia and 1.25% hematocrit
in serum free screening media. Cultures were dispensed at 10 μL
per well using a MultiFlo dispenser (BioTek) and incubated at 37 °C
for 72 h (3% O2, 5% CO2, and 92% N2). A solution of 2.5 μM MitoTracker Red CMXRos and 0.13% saponin
(w/v) was made in screening media and added to each well for 1–2
h at 37 °C to allow for complete red blood cell lysis. The Operetta
(PerkinElmer) high content imaging platform was used to read all 1536-well
plates with the analysis performed by the accompanying Harmony software. Compounds with >50%
inhibition were resourced
from younger stocks and plated in 10-point dose response format. All
screening and analysis steps were then repeated as previously described.
10 nL (final concentration of 2 μM) of each compound was dispensed
using an Echo 550 acoustic dispenser (Labcyte) in 1536-well, black,
clear-bottom plates (ref# 789866-691-2B, Greiner Bio-One). High-content
imaging experiments were performed on stage V gametocytes as previously
described.51 (link) Briefly, mature gametocytes
were diluted to 0.5–0.75% gametocytemia and 1.25% hematocrit
in serum free screening media. Cultures were dispensed at 10 μL
per well using a MultiFlo dispenser (BioTek) and incubated at 37 °C
for 72 h (3% O2, 5% CO2, and 92% N2). A solution of 2.5 μM MitoTracker Red CMXRos and 0.13% saponin
(w/v) was made in screening media and added to each well for 1–2
h at 37 °C to allow for complete red blood cell lysis. The Operetta
(PerkinElmer) high content imaging platform was used to read all 1536-well
plates with the analysis performed by the accompanying Harmony software. Compounds with >50%
inhibition were resourced
from younger stocks and plated in 10-point dose response format. All
screening and analysis steps were then repeated as previously described.
5
Cytotoxicity Assay of Small Molecules
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Rhabdomyosarcoma cell lines Rh30 and Rh6 were seeded in 384-well plates at 2500 cells per well, in 30 µL of media, using a BioTek MultiFlo dispenser. Cultures were grown overnight and then treated with drugs FGF401 (23029), PP1 (14244), Ruxolitinib (11609), and Stattic (14590) that were all purchased from Cayman Chemical. All of the pharmaceuticals were administered at the following concentrations using a D300e digital dispenser (Tecan Trading AG): 0.01, 0.0316, 0.1, 0.316, 1, 3.16, and 10 µM. After 72 h of drug treatment, cultures were treated with an equal volume of CellTiter-Glo reagent (Promega 2020-01-29). Luminescence was measured in a Biotek Instruments Inc. plate reader. The data was then analyzed using Prism GraphPad software.
6
Cryopreserved HEK-AC1 Cell Assay
7
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).
8
High-throughput Cell Viability Screening
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Drugs diluted in dimethyl sulfoxide (DMSO) or water were dispensed at 30-nl volume to 384-well black plates (Corning, #3864) using an Echo 550 acoustic liquid handler (Labcyte). The compounds were plated at five different concentrations in threefold dilutions covering a concentration range relevant for each drug. Cell-killing benzethonium chloride (BzCl, 100 μM) and compound vehicle (DMSO, 0.1%) were used as positive and negative controls, respectively. Cells were diluted to medium at the desired concentration, and the suspension was dispensed to the predrugged plates at 30 μl using a MultiFlo dispenser (BioTek). After 7 days of incubation at 37°C, 10 μl of phosphate-buffered saline (PBS) containing Hoechst (4 μg/ml) and 1/10,000 CellTox Green Dye (Promega) was dispensed per well 1 hour before imaging at a Cytation5 image cytometer or an Opera Phenix (PerkinElmer) confocal screening microscope.
9
Isolation and Culture of Neonatal Rat Ventricular Myocytes
10
Tay-Sachs Fibroblast Calcium Dynamics
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