USP30 inhibitors were dispensed into black, clear bottom, low binding 384 well plates (Greiner) using the ECHO 550 (Labcyte) liquid handler. An amount of 75 nl of compound was dispensed in 100% DMSO. Total reaction volume was 30 µl producing an USP30 inhibitor top concentration of 25 µM. His-tagged, 2× concentrated recombinant human USP30 protein (rhUSP30; amino acids 57–517 of the full-length protein, and a C-terminal 6-His tag, Sf 21 (baculovirus)-derived human USP30 protein) (10 nM; final assay concentration = 5 nM; Boston Biochem) was prepared in USP30 activity assay buffer (50 mM Tris base pH 7.5, 100 mM NaCl, 0.1 mg/ml BSA (Sigma, A7030), 0.05% Tween 20, 1 mM DTT) and 15 µl dispensed into compound containing assay plate using the Multidrop dispenser (Thermo Scientific) and incubated for 30 min at room temperature. Following incubation, 15 µl of 2× concentrated ubiquitin-rhodamine 110 (Ub-Rho110) (200 nM, final assay concentration = 100 nM; Boston Biochem), prepared in USP30 activity assay buffer, was dispensed into the compound-rhUSP30 containing plate and fluorescence immediately read on the FLIPR TETRA plate reader (Molecular Devices). Fluorescence was recorded every 30 s over 1 h and intensity was analysed.
Multidrop dispenser
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multidrop dispenser is a compact, automated liquid handling instrument designed for accurate and precise dispensing of liquids in microplates, tubes, and other labware. It features a 8-channel dispensing head that can deliver volumes ranging from 0.5 to 300 μL. The Multidrop dispenser is a versatile tool for a variety of applications in life science research and laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Enduren, by Promega (5 mentions)
Centro xs960 luminometer, by Berthold Technologies (4 mentions)
Echo 550, by Beckman Coulter (3 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
384 well plate, by Greiner (1 mentions)
Flipr tetra plate reader, by Molecular Devices (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Dharmafect 3 transfection reagent, by Horizon Discovery (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Harmony image analysis software, by PerkinElmer (1 mentions)
Operetta high content imager, by PerkinElmer (1 mentions)
Ethidiumhomodimer 2, by Thermo Fisher Scientific (1 mentions)
Cellcarrier 384 well plates, by PerkinElmer (1 mentions)
Nucview caspase 3 detection reagent, by Sartorius (1 mentions)
Harmony high content imaging software, by PerkinElmer (1 mentions)
Mosquito liquid handler, by SPT Labtech (1 mentions)
Optimem without phenol red, by Thermo Fisher Scientific (1 mentions)
Victor2, by PerkinElmer (1 mentions)
22 protocols using multidrop dispenser
1
High-throughput Screening for USP30 Inhibitors
2
High-Throughput Drug Screening Protocol
3
High-Throughput siRNA Screening for Adipogenesis
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Confluent hASC were trypsinized and reverse transfection was performed using an automated setup (Multidrop dispenser, Thermo Fisher Scientific, Waltham, MA USA) and BioMek plate aspirator/washer (Beckman Coulter, Indianapolis, IN, USA) in 96-well plates. Dharmafect3 transfection reagent (0.35 μL) (GE Healthcare Dharmacon Inc, Lafayette, CO, USA) was mixed with a specific small interfering RNA (siRNA) pool (ON-Target plus, GE Healthcare Dharmacon Inc), reaching a final concentration in cell culture media of 50nM. After 20 minutes of incubation, 10 000 cells in proliferation medium were added to each well in a 96-well plate. A full list of siRNAs containing the order numbers and sequences of the 148 selected transcriptional regulators, nontargeting siRNA (Negative Control) pool, and siRNA targeting the known adipogenic TFs, such as PPARG and CEBPA, are given in Supplemental Table 1 (38 (link)). Each siRNA pool was run in triplicate. PPARG (n = 3) and nontargeting control (n = 6) were included on each test plate (in total 9 plates were utilized for 1 screen). At 24 hours after transfection, medium was changed to differentiation medium and the cells differentiated until day 9 as previously described (37 (link)). In addition to the 148 siRNAs used in the screen, siRNA targeting glucocorticoid receptor (GR) was used for optimization experiments (Supplemental Table 1) (38 (link)).
4
High-Throughput Multiplex Cytotoxicity Assay
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Cells were transferred into CellCarrier 384-well plates (PerkinElmer, Waltham, MA, USA) at a density of 1250 cells/well, using Multidrop dispenser (ThermoFisher Scientific, Waltham, MA, USA). After overnight incubation at 37°C experimental, compounds were added with an ATS Acoustic Transfer System (EDC Biosystems, Fremont, CA, USA). For Ep156T cells the protocol was done in reverse with compounds dispensed before seeding of cells. Fluorescent markers were dispensed in culture medium using ATS system after 72-h compound exposure. Nuclei were stained with cell-permeable Hoechst 33342 (Molecular Probes, Eugene, OR, USA), dead cells with ethidium homodimer-2 (Invitrogen, Carlsbad, CA, USA) and apoptotic cells with NucView caspase-3 detection reagent (Essen Bioscience, Ann Arbor, MI, USA). Cells were imaged with Operetta high-content imager (PerkinElmer, Waltham, MA, USA). Proliferation was measured from the number of nuclei (cells), cell death and apoptosis from positive cells/total cells ratio using Harmony image analysis software (PerkinElmer, Waltham, MA, USA). EC50 values were also assessed with the Harmony software.
5
SARS-CoV-2 S Protein Cell-Cell Fusion Assay
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The DSP assay was performed as described previously (24 (link)). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10-cm plates and incubated overnight (Fig. S1 a and b). Cells were treated with 6 μM EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 h. To test the effect of inhibitor, 0.25 μL of compound library or 1 μL of selected inhibitor dissolved in DMSO was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single-cell suspension (effector and target cells) was added to the 384-well plates by using a Multidrop dispenser (Thermo Fisher Scientific). After incubation at 37°C in 5% CO2 for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Bad Wildbad, Germany).
6
SARS-CoV-2 Entry Inhibition Assay
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For the DSP assay using 293FT cells, effector cells expressing S protein with DSP8-11 and target cells expressing CD26 or ACE2, and TMPRSS2 with DSP1-7 were seeded in 12-well cell culture plates (2 × 105 cells/500 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Scientific, Waltham, MA, USA). After incubation at 37 °C for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Germany). For the DSP assay using Calu-3 or H3255 cells, target cells were seeded in 384-well plates (2 × 104 cells/50 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in DMSO was added to the 384-well plates with 9 μL of culture medium. Next, 40 μL of single cell suspension (effector cells) was added to the wells using a Multidrop dispenser.
7
High-Content Imaging of Autophagy
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HeLa-Difluo™ hLC3 cells (InvivoGen, #heldf-hlc3) were cultivated according to manufacturer’s guidelines. Cells were seeded into 384-well black, clear bottom Biocoat PDL-coated plates (Corning, 356663) with 1250 cells/40 µL using a multidrop dispenser (Thermo Fisher) 24 h prior to treatment. Cells were treated with BafA within a range of 2.7–20 nM. Before live-cell imaging, cells were washed with DPBS (Gibco, 14040-091) containing HOECHST (Invitrogen, H3570) at 3.3 µg/ml final concentration for nuclear staining. Cells were incubated for 15 min at 37 °C. Cells were washed with prewarmed DPBS and transferred to PerkinElmer Operetta CLS live cell chamber at 37 °C and 5% CO2. Images were acquired with a 40 × water-immersion objective with 3 × 3 × 3 images per well for HOECHST, GFP and RFP. Image analysis was performed with Perkin Elmer Harmony high content imaging software. From z-stacks of 1 µm plane height a maximum projection was calculated. After nucleus detection a cytoplasm area around each nucleus was defined using the RFP signal. Within this area, spots were detected for LC3-GFP and LC3-RFP. Features extracted for further data analysis were number of nuclei, size and background corrected spot intensity for GFP/RFP spots.
8
High-Throughput SIRPα Binding Assay
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The assay was conducted as in Table 1 , and all manipulation/incubations were subsequently carried out at room temperature. Optimized reagent amounts for 384 and 1536 well plates are as in Table 2 . DRAQ5-labeled cells were added to 384 or 1536 well plates using a multichannel pipet or a Multidrop dispenser (ThermoFisher). Competing ligand/antibody or control mixed with SIRPα-biotin were added to the cells using a multichannel pipet or Mosquito liquid handler (TTP Labtech). Following NAV488 addition and incubation, cell plates were imaged using the Mirrorball LSC according to Table 1 . For measurements involving fixed cells, the cells were fixed in 4% paraformaldehyde for 30 min on ice, washed and then stained as above.
9
HeLa-Nluc-edit Cells Luminescence Assay
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HeLa-Nluc-edit cells were seeded at a density of 15 000 cells per well into 384 white plates (PerkinElmer, CulturPlate-384, #6007689) in 20 μl of Opti-MEM without phenol red (Thermo Fisher Scientific #11058021) using a multidrop dispenser (Thermo Fisher Scientific) and incubated overnight at 37°C, 5% CO2. The cells were then treated with compounds (see below) for 24 h prior to luminescence signal detection. Briefly, 10 μl of Nluc GLOW Assay reagents (Nanolight Technology # 325) were added and luminescence signal was measured within 10–20 min after the addition using a Victor 2 (Perkin Elmer) multiplate reader at 1 s integration time. Subsequently, 20 μl Fluc Assay reagents (Nanolight Technology # 318) were added to the same wells and luminescence signal was measured with Envision (PerkinElmer) multiplate reader at 0.1 s integration time, within 5–10 min after reagent addition. The ratio between Nluc and FFL signal was calculated for each well and results for each plate were normalized to a negative (0.1% DMSO) and positive (10 μM Nluc inhibitor 1) control. The assay quality was assessed by Z’ factor using the following formula: Z′-factor = 1 − [(3(σp + σn)/(|μp − μn|))], where μn and σn represent the mean and standard deviation of the negative control, and μp and σp represent the mean and standard deviation of the positive control (30 (link)).
10
High-Throughput Drug Screening of Patient-Derived Cells
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Cells were seeded semi-automatically in 384-well plates (Corning) using a Multidrop dispenser (Thermo Fisher Scientific) Patient-derived cells were seeded at a concentration of 1–1.5 × 106 cells/ml, the seeding density of the cell lines are provided in Supplemental Table 2. On the same day, drugs were added to a final concentration of 10 nM, 100 nM or 1000 nM, using the Caliper SciClone ALH3000 liquid handling robot. The following commercially available drug libraries were utilized: Prestwick Chemical library (Prestwick Chemical, France), anti-neoplastic sequoia library (Sequoia Research Products, United Kingdom), Epigenetics library (Enzo Life Sciences, Belgium), Epigenetics screening library (Cayman Chemical, MI, USA), Spectrum collection (Microsource, CT, USA), the Cell cycle/DNA Damage compound library (MedChemExpress, Sweden), and some additional compounds (Supplemental Table 3). Upon drug exposure, cell viability was assessed by a 4-day thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) assay [13] (link).
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