MEF cells were plated overnight at 2 500 cells per well and 2 000 cells per well in CellCarrier-384 Ultra microplates (Perkin Elmer). Cells were distributed under 30 μl of medium. Next day plates were centrifuged before and after compounds adding at 900 rpm, 5 minutes, 20°C. Parthenolide was added with an ECHO 550 (Labcyte) in dose response from 1.7 to 50 μM in duplicate. After parthenolide, paclitaxel was added too in half microplates with ECHO 550 (Labcyte) at a final concentration of 5 μM. Compounds were incubated on cells 1h, 4h and 24h, then plates were fixed and labeled.
Echo 550
Manufactured by Beckman Coulter
Sourced in United States, Ireland
The Echo 550 is a liquid handling instrument designed for precise and accurate sample transfer. It utilizes acoustic energy to aspirate and dispense liquids without the need for physical contact, enabling non-destructive processing of samples. The Echo 550 is capable of transferring a wide range of volumes, from nanoliters to microliters, with high reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
Multidrop combi reagent dispenser, by Thermo Fisher Scientific (6 mentions)
Celltiter glo, by Promega (6 mentions)
Sypro orange, by Thermo Fisher Scientific (5 mentions)
Multiflowfxliquid dispenser, by Agilent Technologies (4 mentions)
Pherastar, by BMG Labtech (4 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (4 mentions)
Evos fl auto 2, by Thermo Fisher Scientific (3 mentions)
Cfx384 real time pcr instrument, by Bio-Rad (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Multidrop dispenser, by Thermo Fisher Scientific (3 mentions)
384 well plate, by Corning (3 mentions)
Echo qualified 1536 well source plates, by Beckman Coulter (3 mentions)
Viewlux imaging plate reader, by PerkinElmer (3 mentions)
Opera phenix, by PerkinElmer (3 mentions)
510 confocal microscope, by Zeiss (2 mentions)
Clariostar, by BMG Labtech (2 mentions)
Multidrop combi mdc, by Thermo Fisher Scientific (2 mentions)
Biomek fx, by Beckman Coulter (2 mentions)
127 protocols using echo 550
1
Cell Viability Assay with Parthenolide and Paclitaxel
2
High-Throughput Screening for SARS-CoV-2 Helicase Inhibitors
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The screen was performed in 384-well Greiner black flat-bottom plates (Greiner 781076). A custom compound library containing over 5000 compounds assembled from commercial sources (Sigma, Selleck, Enzo, Tocris, Calbiochem, and Symansis) were distributed in 24 plates using an Echo 550 (Labcyte) across column 3–22; 2.5, or 12.5 nl of a 10 mM stock of the compounds dissolved in DMSO were arrayed and pre-dispensed into the assay plates using an Echo 550 (Labcyte), before being sealed and stored at −80°C until screening day. All 384 wells on the plates contain 1 µl DMSO. First, 10 µl 2× Nsp13 mix (6 nM FH-nsp13) in HTS assay buffer (20 mM HEPES pH 7.6, 20 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.1 mg/ml BSA) was dispensed into columns 2–23 of the plates to incubate with the compounds for 10 min at room temperature. Then 10 µl 2× substrate mix (200 µM ATP, 360 nM DNA substrate, 1800 nM competitor) in HTS assay buffer was dispensed to start the enzymatic reaction. The plates were then spun briefly and transferred to a Tecan microplate reader to monitor changes in fluorescence signal in a kinetic mode. The fluorescence signal was first read at 2 min of reaction and then read every 1.5 min for 10 cycles. The screening for nsp13 inhibitors was done twice, one with a final compound concentration of 1.25 µM and one with 6.25 µM.
3
High-throughput drug response screening in primary cells
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MNCs were added to pre-spotted drug plates (FIMM HTB)28 (link) or custom plates made with an Echo 550 (Beckman Coulter, Brea, CA, Supplementary Table 8 ). Cells were incubated in complete RPMI for 72 h (37 °C, 5% CO2). Cell viability was measured by CellTiterGlo (CTG, Promega, Madison, WI) on an EnSight plate reader (PerkinElmer, Waltham, MA). Drug sensitivity scores (DSS) were calculated with Breeze29 (link). Selective DSS (sDSS) values were calculated by subtracting healthy BM controls DSS values from patient DSS values. Fold growth was measured in untreated cells as the ratio of luminescence signal at 0 h and 72 h.
4
High-Throughput SARS-CoV-2 Pseudovirus Assay
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Using an ECHO550 liquid handler (Beckman Coulter), 40 nL of the stock compound (10 mM in 100% DMSO) was added to the Greiner bio-one 384-wells plate (40 μL final volume, final concentration 10 μM). Then, 100 μL (containing about 17,000 cells) of each cell suspension (Vero/cSLAM-GFP/LgBiT and Vero-Fs-sH-RFP/HiBiT) was seeded to the wells using a Multidrop Combi Reagent Dispenser (Thermo Scientific, Waltham, MA, USA) and incubated at 37 °C for 24 h. After the incubation, corresponding substrate (Nano-Glo® Live Cell Assay System, Promega) was added to the plate using Biotek Microflo Select Dispenser (Agilent) and the total amount of luminescence was measured by Synergy NEO HTS Multi-Mode Microplate Reader (Biotek). Afterwards, all the statistical analysis was done by the BSF using the internal Laboratory Information Management System (LIMS).
5
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)].
6
Sustainability Logo Encoding Protocol
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The digital image of a sustainability
logo (57 × 57 pixels) was encoded using a Rate 1/3 Turbo code,
multiplied by a permutation matrix, concatenated with the training
set, and converted into a one-dimensional binary vector. The training
spots are a set of 27 = 128 enumerated binary mixtures,
which is represented in the binary vector as 128 × 7 = 896 bits.
The assembled one-dimensional (1-D) vector was reshaped into an M × N matrix, where M represents the number of waste mixtures and N represents
the number of independent mixtures. For each true (“1”)
value in the matrix, 30 nL (12 2.5 nL droplets) of the mth waste mixture was transferred from the 384-well library plate
to the nth location on a metal MALDI plate using
an acoustic liquid handler (Echo 550, Beckman Coulter). This implies
that each location was a mixture of up to 7 library elements and contained
up to 210 nL. Once all transfers are complete, the data plate was
left to dry in a fume hood overnight. The resulting dried mixture
spots were typically 1 mm in diameter.
logo (57 × 57 pixels) was encoded using a Rate 1/3 Turbo code,
multiplied by a permutation matrix, concatenated with the training
set, and converted into a one-dimensional binary vector. The training
spots are a set of 27 = 128 enumerated binary mixtures,
which is represented in the binary vector as 128 × 7 = 896 bits.
The assembled one-dimensional (1-D) vector was reshaped into an M × N matrix, where M represents the number of waste mixtures and N represents
the number of independent mixtures. For each true (“1”)
value in the matrix, 30 nL (12 2.5 nL droplets) of the mth waste mixture was transferred from the 384-well library plate
to the nth location on a metal MALDI plate using
an acoustic liquid handler (Echo 550, Beckman Coulter). This implies
that each location was a mixture of up to 7 library elements and contained
up to 210 nL. Once all transfers are complete, the data plate was
left to dry in a fume hood overnight. The resulting dried mixture
spots were typically 1 mm in diameter.
7
High-Throughput Screening of PDAC Therapeutics
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PDAC PCOs were plated in a 96-well assay (as above), including 5000 cells in a 10 µL matrix. A custom library of 80 lead therapeutic agents was identified from ongoing phase I/II clinical trials in development. These agents were prepared per the manufacturing guidance (MedChemExpress, Princeton, NJ, USA) and dosed at physiologic values up to 5 μM (Table S3 ). Small molecules were added using Echo 550 (Beckman Coulter, Indianapolis, IN, USA) for low-volume liquid handling in collaboration with the UWCCC Drug Development Core. No pharmacologic adjustments or media replacements were performed over the duration of therapeutic screening. Following drug incubation, cell viability was measured by adding 3D Cell Titer-Glo luminescence 33.3% v/v (Promega, Madison, WI, USA). After adding the reagent, the plate was shaken for 5 min, incubated for 25 min at room temperature in the dark, and the luminescence was measured using a Gen5 plate reader (Biotek, Santa Clara, CA, USA).
8
Luminescent HTS Assay for Mtb PheRS Inhibitor Discovery
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This assay used for HTS was based on Kinase-Glo luminescent Kit (Promega). The application with aaRS was previously descried (27 (link), 45 (link)). Screening was carried out in Corning 384-well white flat bottom microplates (catalog 3570). The final concentrations in the reaction are 25 mM Hepes pH 7.4, 140 mM NaCl, 40 mM MgCl2, 30 mM KCl, 1 mM tris(2-carboxyethyl)phosphine, 0.1 mg/ml bovine serum albumin, and 0.004% Tween-20. Compounds from Selleck-2148 bioactive library (Selleckchem) 10 mM stock were prepared in assay plate using Echo 550 (Labcyte) to final concentration of 50 μM. The 50-nL compound volume is negligible in each reaction. Dimethyl sulfoxide (DMSO) was added into negative control wells, and tool compound GDI05-001 was added in positive control wells. About 5 μl Mtb PheRS in Hepes buffer was added to 100 nM using Multidrop dispensers (Thermo), and the compound–enzyme mixture was incubated for 30 min. Then 5-μl substrate mixture (1 μM ATP, 20 μM l -Phe, and 0.1 mg/ml tRNAPhe) was added to start the reaction at 37 °C and incubated for 2 h. After cooling down the plates, 10-μl diluted Kinase-Glo Max reagent (1/50 diluted with buffer 50 mM Tris pH 7.5, 5% glycerol) was added and incubated for 15 min. Luminescence was recorded using SpectraMax M5 (Molecular Devices).
9
Multivariate Gene Expression and Immunofluorescence Analyses
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Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). A 2uL PCR reaction was set up using acoustic liquid handler (ECHO 550, Labcyte) and performed with the CFX384 real-time PCR instrument (Bio-Rad). Assays included at least three technical and two biological replicates unless otherwise stated, and were run using the Ssoadvanced Universal SYBR Green Supermix (Bio-Rad).
For immunofluorescence staining, cells were fixed with 4% paraformaldehyde, and simultaneously permeabilized and blocked with 0.2% Triton X-100 + 5% BSA +5% normal goat serum (or a serum specific to the host of secondary antibody) for an hour. Cells were then incubated with appropriate dilution of primary antibodies (Anti-PDGFR alpha (1:200); PE anti-human CD140a (1:100); Anti-SOX10 (1:100); Anti-NKX2.2 (1:50); Anti-MBP (1:100); Anti-OLIG2 (1:500); Anti-O4 (1:200)) overnight followed by secondary antibodies (Alexa Fluor 488/647 (1:500)) for 2 hours. Fluorescence images were taken using either the EVOS FL Auto 2 (ThermoFisher Scientific) or Zeiss 510 confocal microscope.
For immunofluorescence staining, cells were fixed with 4% paraformaldehyde, and simultaneously permeabilized and blocked with 0.2% Triton X-100 + 5% BSA +5% normal goat serum (or a serum specific to the host of secondary antibody) for an hour. Cells were then incubated with appropriate dilution of primary antibodies (Anti-PDGFR alpha (1:200); PE anti-human CD140a (1:100); Anti-SOX10 (1:100); Anti-NKX2.2 (1:50); Anti-MBP (1:100); Anti-OLIG2 (1:500); Anti-O4 (1:200)) overnight followed by secondary antibodies (Alexa Fluor 488/647 (1:500)) for 2 hours. Fluorescence images were taken using either the EVOS FL Auto 2 (ThermoFisher Scientific) or Zeiss 510 confocal microscope.
10
EZH2 histone methyltransferase assay
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The EZH2 substrate (0.05 mg/mL core histone) was added in the freshly prepared reaction buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO). The PRC2 complex (complex of human EZH2, human EED, human SUZ12, human AEBP2, and human RbAp48) was delivered into the substrate solution and the mixture was mixed gently. Afterwards, the tested compounds dissolved in DMSO were delivered into the enzyme/substrate reaction mixture by using Acoustic Technology (Echo 550, LabCyte Inc. Sunnyvale, CA, USA) in nanoliter range, and 3H-SAM was added into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 1 h at 30 °C and then it was delivered to filter-paper for detection. The data were analyzed using Excel and GraphPad Prism software for IC50 curve fits. The experiments were performed in duplicate.
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