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Related product: D300e Digital Dispenser
Two days after seeding, organoids were 605 treated with ERKi SCH772984 (0 nM to 10 ÎźM) or MEKi AZD6244 (selumetinib) (0 to 2.5 ÎźM), randomized on a Tecan D300e drug dispenser. O
Read moreTitrations of BodipyFL-E7820 (4) were carried out by mixing 200 nM biotinylated Strep-II-Avi-tagged DCAF15 variants or an equivalent volume of the assay buffer, 2 nM terbium-coupled streptavidin in the same assay buffer. After dispensing the assay mixture, an increasing concentration of BodipyFL-E7820 (4) was dispensed in the 384-well plate using a Titrations of BodipyFL-E7820 (4) were carried out by mixing 200 nM biotinylated Strep-II-Avi-tagged DCAF15 variants or an equivalent volume of the assay buffer, 2 nM terbium-coupled streptavidin in the same assay buffer. After dispensing the assay mixture, an increasing concentration of BodipyFL-E7820 (4) was dispensed in the 384-well plate using a Titrations of BodipyFL-E7820 (4) were carried out by mixing 200 nM biotinylated Strep-II-Avi-tagged DCAF15 variants or an equivalent volume of the assay buffer, 2 nM terbium-coupled streptavidin in the same assay buffer. After dispensing the assay mixture, an increasing concentration of BodipyFL-E7820 (4) was dispensed in the 384-well plate using a D300e digital dispenser normalized to 2% DMSO and then incubated for 15 min at room temperature. normalized to 2% DMSO and then incubated for 15 min at room temperature. normalized to 2% DMSO and then incubated for 15 min at room temperature
Read moreDoxycycline (from a 0.3% Tween stock solution) was added to the remaining cells where applicable and drugs (from DMSO solutions) were added using an HP D300 Digital Dispenser
Read moreAbout 24 h later, drugs were added at the indicated concentrations using the HP D300 Digital Dispenser (HP)
Read moreThe drugs and their combinations were added 1 h after plating the organoids using the Tecan D300e Digital Dispenser. Drugs were dispensed in a randomized manner and DMSO end concentration was 1% in all wells. 120 h after adding the drugs, ATP levels were measured using the Cell-Titer Glo2.0 (Promega BV) according to the manufacturerâs instructions and luminescence was measured using a SpectraMax microplate reader (Molecular Devices). Results were normalized to vehicle (DMSO = 100%) and baseline control (navitoclax 20 ÂľM)
Read more384-well BioCoat Collagen I (Corning) microtiter plates at a density of 1200, 1800, 2000, 1600, and 1200 cells per well, respectively. The following day, compound or DMSO was added to wells using an HP D300 digital dispenser instrument. Each compound was tested using 9 concentrations, in quadruplicate (four wells treated in parallel). Cell viability was assayed 6 d after compound addition with th;384-well BioCoat Collagen I (Corning) microtiter plates at a density of 1200, 1800, 2000, 1600, and 1200 cells per well, respectively. The following day, compound or DMSO was added to wells using an HP D300 digital dispenser instrument. Each compound was tested using 9 concentrations, in quadruplicate (four wells treated in parallel). Cell viability was assayed 6 d after compound addition with th
Read moreFor IC50 measurement using the FGFR inhibitors, cells were dissociated into single cells and seeded into a 384-well tissue culture plate, each well with 200 viable cells and 40 ÎźL of growth medium. After 24 hours, compounds were added to each well over a 15-point concentration range, along with DMSO controls, using a Tecan D300e digital drug dispenser. Cells were cultured for 5 days in the presence of compound before assessing viability by adding 15 ÎźL of CellTiterGlo (Promega) to each well, incubating for 20 minutes at room temperature on a shaker, and measuring luminescence using an Envision
Read moreThe drugs were added 1 hour after plating the organoids using the Tecan D300e Digital Dispenser (Tecan). Nutlin-3 (Cayman Chemical, catalog no. 10004372), Niraparib (Selleckchem, catalog no. S2741), AZD4547 (ApeXbio, catalog no. A8250), everolimus (LC Laboratories, catalog no. E4040), vemurafenib (Selleckchem, catalog no. S1267), and alpelisib (LC Laboratories, catalog no. A4477) were dissolved in DMSO. Cisplatin (Sigma, catalog no. C2210000), carboplatin (Sigma, catalog no. C2538), and cetuximab (obtained from hospital pharmacy) were dissolved in PBS containing 0.3% Tween-20, which was required to dispense these drugs using the HP printer. All wells were normalized for solvent used. DMSO percentage never exceeded 1%, PBS/Tween-20 percentage never exceeded 2%. Drug exposure was performed in triplicate for each concentration shown. For a layout of the drug screen, see Supplementary Fig. S12
Read moreAfter 24 h, the CCS1477 and HATi329 compounds were added in 4 replicates using a HP D300 digital dispenser (HP), with 12 wells of DMSO-treated control cells. CellTiter-Glo readings were acquired after 48 h using a Tecan Infinite 200 plate reader. T
Read moreWe analyzed 31 therapeutic compounds on the whole panel of 34 LCCLs (Supplementary Table 4). Drug screening was performed using the HP D300 digital dispenser (Tecan, Mannedorf, Switzerland) to create dose-response curves as described in the Supplementary
Read moreand oxaliplatin (oxaliplatin Cmax in patients = 3.8 to 10.1 mM; oxaliplatin range in vitro = 0.319 to 200 mM) in patients using a Tecan D300e digital dispenser (31â33)
Read moreHTS dispenser (TTP Labtech). For single-drug dose response studies, cells were seeded into 96-well plates at 300 000 cells/mL, and the compounds were added using a D300e digital dispenser (Tecan). Cell proliferation was
Read moreBAX is a critical effector of the mitochondrial cell death pathway in response to a diverse range of stimuli in physiological and disease contexts. Upon binding by BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation, resulting in mitochondrial outer ;ed with indicated concentrations of BTSA1 and BAI1 or DMSO in no FBS media for 2.5 hr, followed by 10% FBS replacement to a final volume of 25 Îźl. Dilutions of compounds were performed using a TECAN D300e Digital Dispenser from 10 mM stocks. Cell viability was assayed at 6 hr by addition of CellTiter-Glo according to the manufacturerâs protocol (Promega), and luminescence measured using a F200
Read moreor 1h. Then, e-Coelenterazine Prolume Purple (Methoxy e-CTZ; Nanolight) was added to a final concentration of 2 ÎźM followed immediately by the addition of increasing agonist concentrations using the HP D300 digital dispenser (Tecan). After 10 min of incubation at room temperature, the BRET signal was subsequently detected at 0.2 sec integration time using a Synergy NEO plate reader (BioTek) equip
Read moreCompounds were solubilized in DMSO and dispensed into 384-well plates using a D300e Digital Dispenser (HP)
Read morecin (100 Âľg mLâ1) and puromycin (10 mg mLâ1). BRET assays Ligand preparation: Agonists were dissolved in DMSO and spotted on 96-well white bottom microplates (Greiner bio-one) using the HP D300 Digital Dispenser (Tecan Life Sciences). DMSO concentration was normalized for each point at 0.334%. GÎąi and GÎąo-activation assay: HEK 293 were co-transfected with DOR or MOR (human or rat)
Read more384-well plates with 50 ÎźL FluoroBrite DMEM media (Thermo Fisher Scientific A18967) containing 10% FBS per well a day before compound treatment. Compounds and 100 nM dBET6 were dispensed using a D300e Digital Dispenser (HP), normalized to 0.5% DMSO, and incubated with cells for 5 h. The assay plate was imaged immediately using an Acumen High Content Imager (TTP Labtech) with 488 nm and 56;384-well plates with 50 ÎźL FluoroBrite DMEM media (Thermo Fisher Scientific A18967) containing 10% FBS per well a day before compound treatment. Compounds and 100 nM dBET6 were dispensed using a D300e Digital Dispenser (HP), normalized to 0.5% DMSO, and incubated with cells for 5 h. The assay plate was imaged immediately using an Acumen High Content Imager (TTP Labtech) with 488 nm and 56
Read morerimental details: Cell lines were seeded into 384-well, white-walled, clear bottom plates at a density of 500 cells/well. Twenty-four hours after seeding, combination drugs were administered using an HP D300 Digital Dispenser in matrix format. Drug was administered such that the final volume of DMSO did not exceed 0.5%. The cells were then incubated for seven days and cell luminescence was measure
Read morells were seeded in triplicate on 96-well plates (Corning, Falcon) at 0.5â1 Ă 104 cells per well and treated with gradient concentrations of compounds diluted in DMSO (Dimethyl sulfoxide) using HP D300 Digital Dispenser (Tecan). Three compounds were used KIN001-102/Akt Inhibitor VIII (124018, Merck), 681640/Wee1 Inhibitor (681640, Merck) and a ROCK1 inhibitor, GSK269962A (S7687, Selleckchem)
Read moreof HKTx dispensed into 384âwell nonâbinding, low volume, HiBase, black microplates (greiner bioâone). Note, all FICD variants were dispensed directly into the microplate well solutions using a D300e digital dispenser (Tecan). The plate was then sealed and the final 20 Îźl (Fig 6F) or 15 Îźl reactions (Fig 6G) were incubated for 45 min at 10°C, whilst shaking at 300 rpm on a ThermoMix
Read more04 pGEMâT Easy vector syst. I Promega A3600 Nanodrop Lite Thermo Fisher TruSeq Nano kit Illumina 20015965 Illumina HiSeqX Illumina DNeasy blood & tissue kit Qiagen 69506 D300e Digital Dispenser Tecan Micromanipulator Narishige Mâ152 Microinjector Narishige IMâ5B Stereomicroscope Leica MZ75 FlexMAP3D BioâRad Laboratories Methods and Prot;ted by pipetting before being resuspended in 5% BME/AO media (15â20,000 organoids mlâ1) and dispensed into 384âwell microplates (Greiner). Each compound was dispensed at 1â100 ÎźM using a TECAN D300e Digital Dispenser, and after 5 days, cell viability was assayed as described above. Paclitaxel (T7402), methotrexate (A6770), crizotinib (PZ0240), and cisplatin (C2210000) were obtained;04 pGEMâT Easy vector syst. I Promega A3600 Nanodrop Lite Thermo Fisher TruSeq Nano kit Illumina 20015965 Illumina HiSeqX Illumina DNeasy blood & tissue kit Qiagen 69506 D300e Digital Dispenser Tecan Micromanipulator Narishige Mâ152 Microinjector Narishige IMâ5B Stereomicroscope Leica MZ75 FlexMAP3D BioâRad Laboratories Methods and Prot
Read moreFor TK-216, cell lines were seeded in 384-well plates at the density of 2,000 cells/well using the VIAFLO 96/384 hand-held electronic channel pipette (Integra Biosciences AG) and then treated with D300e Digital Dispenser (Tecan)
Read moreCapmatinib was then added from a 10 mM dimethyl sulfoxide (DMSO) stock solution using a HP D300 Digital Dispenser (Tecan)
Read moreCellTiterGlo (Promega) reagent was added 96 hours after beginning treatment, and luminescence was measured on a Spectramax M5 spectrophotometer (Molecular Devices). All conditions were tested as triplicate biological replicates. All drug treatments were administered using a Tecan D300e Digital Dispenser. GI50 were calculated using GraphPad Prism Software using the function log (inhibitor) versus response-variable slope (4 parameters). Values are capped at the maximum drug dose used
Read more3,000 cells per well were seeded using a microplate Dispenser (MultiFloâ˘, BioTek) in 384-well clear bottom, black wall plates (Corning). Drugs were added using the Tecan D300e digital dispenser (Tecan). After 72-h incubation, alamarBlue⢠cell viability reagent (ThermoFisher) was added and viability was quantified by measuring fluorescence in a plate reader (Tecan Infinite m1000 pro, Ex: 530 nm; EÎť: 590). Synergy analysis was conducted using Compusyn software69
Read moreSkip to article ;Using an automated liquid-handling robot such as the HP D300e Digital Dispenser, it is possible to dispense compounds directly into microtiter plates in an arbitrary pattern, randomizing the locations of control and technical replicates and converting systematic error into ;Using an automated liquid-handling robot such as the HP D300e Digital Dispenser, it is possible to dispense compounds directly into microtiter plates in an arbitrary pattern, randomizing the locations of control and technical replicates and converting systematic error into
Read moreMaster drug stocks were stored at 10 mM in DMSO for TKIs and 2 M in water for 2DG with 0.03% Tween 20. Tween was added to facilitate dispensing of drugs in aqueous solvent by the HP D300 dispenser
Read moreSigma- Aldrich). Drugs (Supplemental Table 1) were purchased from Selleckchem and added to the cells using the HP D300 Digital Dispenser (Tecan). 24h after seeding cells, medium was changed to 19 Îźl culture medium
Read moreell) in 100 mL complete medium and treated using a TECAN D300e digital dispenser (HP). After 72 hours of treatment, relative cell viability was assessed by measuring the ATP content in each well. Therefore, CellTiter-Glo Reagent (Promega, catalog no. G7573) was added 1:1. Upon 10 minutes of incubation, luminescence intensity was measured with a plate reader (Tecan Infinite M1000 Pro) and normalized to intensities of control wells
Read moreAll drugs were dispensed by a HP-D300 automated liquid dispenser (TECAN). Samples were incubated for 6 days at 37 °C, and cell viability was measured by CellTiter-Glo 3D kit (Promega) on a SpectraMax M5e (Molecular Devices). Cell viability measurements were performed in duplicate wells for each clone. Survival ratios in drug-treated organoids were normalized to the average survival in a DMSO control
Read moreThen, 21 concentrations of afatinib, gefitinib, pictilisib, GDC-0068, AZD8055, everolimus (all Selleckchem), or tamoxifen (Sigma) as well as DMSO controls were added in duplicate using a Tecan D300e digital dispenser (Life Sciences). To measure ATP as proxy for viable cells, 40 ÎźL of CellTiter-Glo 3D Reagent (Promega) per well were added after five days, before shaking the plate for 30 min at room temperature and reading luminescence on a SpectraMax microplate reader (Molecular Devices)
Read moreIndicated cells were cultured and seeded into 96-well plates at a density of 500â2,000 cells per well, depending on growth rate. Twenty-four hours later, drugs were added at the indicated concentrations using the HP D300 Digital Dispenser (HP). Cells were imaged every 4 h in IncuCyte ZOOM (Essen Bioscience). Phase-contrast images were analyzed to detect cell proliferation based on cell confluence
Read moreFor dose-response curves and viability assays in melanoma, 1 Ă 103 cells were plated in 96-well plates and ADC was added 3 h after seeding with the HP D300 Digital Dispenser (Tecan)
Read moresynergy between the different compounds, ALL-SIL, mouse leukemic cells or patient-derived xenograft cells were seeded into 96-well plates and the compounds were added in a randomized fashion using a D300e digital dispenser (Tecan). The DMSO concentration was normalized. Cell proliferation was measured after 48 hours using the ATPlite luminescence system (PerkinElmer) using a Victor multilabel pla;synergy between the different compounds, ALL-SIL, mouse leukemic cells or patient-derived xenograft cells were seeded into 96-well plates and the compounds were added in a randomized fashion using a D300e digital dispenser (Tecan). The DMSO concentration was normalized. Cell proliferation was measured after 48 hours using the ATPlite luminescence system (PerkinElmer) using a Victor multilabel pla
Read morein PBS and stained with 0.1% crystal violet in water. Incucyte proliferation assays were carried out in 96-well plates at a density of 500â1000 cells per well. 24 h later, drugs were added using HP D300 Digital Dispenser (HP) at indicated concentrations. Cells were imaged every 4 h in IncuCyte ZOOM (Essen Bioscience). Phase-contrast images were collected and analyzed to detect cell prolifer
Read moreIn each of these experiments, an inoculum of 104 cells was inoculated into 150 Îźl of media in a Nunc 96-well flat-bottom microplate. Antibiotics were added into the wells as indicated using a D300e digital dispenser (Tecan), which dispenses a nanolitre-scale volume of each antibiotic. Each concentration combination was performed in duplicate wells. Multiple untreated control wells (no antibiotics) were designated on each plate (2â6% of the wells in each experiment). To avoid systematic positional effects across the plates, the wells chosen for each drug combination on the plates were randomized. The plates were incubated at 30 °C with shaking (Liconic orbital shaker STX44), and OD600 nm was measured at least every 25 min using a Tecan robotic system and the Infinite M200 Pro reader. To enhance uniformity, the plate orientations in the shaker were rotated 180° following every measurement
Read moree IKZF1 homology model was taken from17. Time-resolved fluorescence resonance energy transfer (TR-FRET) Compounds in dimerization assays were dispensed in a 384-well microplate (Corning, 4514) using D300e Digital Dispenser (HP) normalized to 2% DMSO into 200 nM biotinylated His6-avi-bromodomain (WT or mutant, see Figure legends) or 80 nM biotinylated StrepII-avi-IKZF1Î (See Figure legends), 100;e IKZF1 homology model was taken from17. Time-resolved fluorescence resonance energy transfer (TR-FRET) Compounds in dimerization assays were dispensed in a 384-well microplate (Corning, 4514) using D300e Digital Dispenser (HP) normalized to 2% DMSO into 200 nM biotinylated His6-avi-bromodomain (WT or mutant, see Figure legends) or 80 nM biotinylated StrepII-avi-IKZF1Î (See Figure legends), 100
Read morecytometry-based bioassays CEMTART tat/rev++ cells were maintained as described for cytoprotection assays. Drugs suspended in DMSO were added to media (100 Âľl) in 96-well flat-bottom plates using an HP D300 Digital Dispenser. All wells were normalized with DMSO to 0.5% total volume. Divamide stock concentrations of either 5 or 2 Âľg/Âľl were utilized for assays employing a concentration range sui
Read moren 384-well plates and incubated overnight. Next, cell lines and organoids were co-treated with three-fold dilution series of JQ1 (5.1 nMâ33.3 ÎźM) and THZ1 (0.051 nMâ0.333 ÎźM), using the HP D300 Digital Dispenser (Tecan). Control cells were treated with DMSO. Cell viability was determined after 72 h (classical cell lines) or 120 h (organoids) using the 3-(4.5-dimethylthiazol-2-yl)
Read moreDrug susceptibility was measured using the SYBR Green I method as previously described44 [/articles/s41467-018-04104-z#ref-CR44]. In brief, tightly synchronized 0â6 h rings were grown for 72 h in the presence of different concentrations of drugs in 384-clear-bottom well plates at 1% hematocrit, 1% starting parasitemia and 40 Îźl of 0.5% Albumax culture media. Growth at 72 h was measured by SYBR Green I (Lonza, Visp, Switzerland) staining of parasite DNA. A 24-point dilution of PPQ (Sigma-Aldrich, St. Louis, MO) and a 12-point dilution series of the rest of the drugs (DHA, ART, AS, MEF, and LUM; Sigma-Aldrich, St. Louis, MO) were carried out in triplicate and repeated with at least three biological replicates. Drug stocks were resuspended in dimethyl sulfoxide except for CQ prepared in 0.1% Triton X-100 in water and PPQ prepared in 0.1% Triton X-100 and 0.5% lactic acid in water to ensure complete dissolution, as lactic acid enhanced PPQ solubility45 [/articles/s41467-018-04104-z#ref-CR45]. Drugs dispensed by a HP D300 Digital Dispenser (Hewlett Packard Palo Alto, CA), with the Triton X-100 enhancing dispensing of aqueous drug stocks. Relative fluorescence units was measured at an excitation of 494 nm and emission of 530 nm on a SpectraMax M5 (Molecular Devices Sunnyvale, CA) and analyzed using GraphPad Prism version 7 (GraphPad Software La Jolla, CA). EC50 values were determined with the curve-fitting algorithm log(inhibitor) vs. responseâVariable slope except for PPQ. PPQs bimodal doseâresponse would not allow for any curve-fitting hence susceptibility was measured by calculating the area under the second response curve (AUC). AUC was calculated by fitting a six-dimensional least-square polynomial equation to each sampleâs doseâresponse curve and integrating the fitted equation over the region corresponding to the second response curve. These equations were fit to the data using the polyfit module in NumPy, a fundamental package for scientific computing using Python. We chose to use a six-dimensional least-square polynomial equation because it captures the dynamics of the second response curve better than equations with fewer dimensions. Using equations with dimensions > 6 do not result in major differences in calculated AUC values. In fact, AUC values calculated using the boundaries identified by our equation fitting method (0.10 ÎźM â 30 ÎźM) gave roughly the same result. The boundaries of the second response curve were determined by identifying the drug concentration corresponding to the average local minima before and after the second response curve across all drug-assayed samples
Read moreed in this study were provided by Eli Lilly as 10 mM stocks in DMSO, in heat-sealed 96-well plates or purchased from Tocris and dissolved in DMSO (Sigma Catalog Number D5879, Lot Number SHBF7682V). HP D300 T8 cassettes for pilot experiment were from Tecan. All other automated liquid handling consumables, experiments in stages 1 and 2, were from Perkin Elmer. Concentration of each drug was select;ed in this study were provided by Eli Lilly as 10 mM stocks in DMSO, in heat-sealed 96-well plates or purchased from Tocris and dissolved in DMSO (Sigma Catalog Number D5879, Lot Number SHBF7682V). HP D300 T8 cassettes for pilot experiment were from Tecan. All other automated liquid handling consumables, experiments in stages 1 and 2, were from Perkin Elmer. Concentration of each drug was select
Read more, 3.8 ÂľM TGF-Îą, 1.2 ÂľM IL-6, 0.9 ÂľM TNF-Îą, 0.9 ÂľM IFN-Îł, and 4.0 ÂľM LPS) with and without the addition of 5 ÂľM GSK126. After 24 h, BTZ was added in a dosage range of 0.01 to 300 nM using the TECAN d300e compound printer, including four replicates per dosage condition with randomized well distribution and DMSO normalization. GSK126 treatment was renewed 24 h after the application of BTZ us
Read moreIn the case of the validation experiments with HCC1806 and HCC1937 cell lines expressing 4E-BP1 or GFP constructs, cells were treated in a 9-point 1:3 dilution series of AZD8055, BEZ235, or GDC0941 using a HP D300 Digital Dispenser (HewlettPackard), while all other experimental conditions remained the same. Each assay was carried out in biological triplicate. Each replicate of a doseâresponse experiment was further analyzed by normalization to the negative and positive control (the normalized data are provided in Supplementary Table S3) and fitting to a four-parameter sigmoid function that allowed for the calculation of the IC50 (dose at which viability is 50% of the untreated control). The IC50 estimates are provided in Supplementary Table S4. For model inference, full doseâresponse curve data were used
Read morewere distributed into six 96-well plates, with 12 individuals per plate exposed to a concentration (0, 0.03, 0.1, 0.3, 1, 3, 5, or 10ÎźM) using a 20-mM stock of Abamectin digitally dispensed using an HP D300e (Hewlitt Packard HP D300e). Second, a narrower range including 0, 0.5, 0.7, and 0.9ÎźM was performed using 192 individuals (48 per concentration). From these data, the final critical concentr;First, 576 individual zebrafish embryos were distributed into six 96-well plates, with 12 individuals per plate exposed to a concentration (0, 0.03, 0.1, 0.3, 1, 3, 5, or 10 Îź M ) using a 20 -mM stock of Abamectin digitally dispensed using an HP D300e (Hewlitt Packard HP D300e)
Read moreCompounds were dissolved in DMSO to 20 mM and a digital dispenser HP D300 (Tecan) was employed to set up the concentration response experiments. Prior to the analysis of compound binding, three running buffer blanks were injected to equilibrate the instrument
Read moreSW620 cells were seeded in white 96-well plates (Greiner) at a density of 2000 cells/well and allowed to attach for 24 hr. 5-FU and doxorubicin were prepared in DMSO and dispensed using an HP D300 compound dispenser
Read moreDilutions of compounds was performed using a TECAN D300e Digital Dispenser from 10 mM stocks
Read morem inhibitory concentration for each drug is given in Table 1. Assays were performed in clear, flat-bottom 384-well microplates by dispensing nanoliter volumes of drugs using a digital drug dispenser (D300e Digital Dispenser, HP). Dispense locations were randomized within each plate using the software to minimize the plate position effect. Mid-log phase cultures were diluted to an optical density (
Read moreThe cells were incubated for 90 min in a humidified atmosphere at 37 °C and 5% CO2. Compounds in DMSO stocksolutions were added using a HP D300 Digital Dispenser (Tecan, Männedorf, Switzerland)
Read moretion plates and a Seiko pin transfer robot system. Immediately after, A375 and WM115 cells were then treated with 1 ÎźM vemurafenib and COLO858 cells were treated with 0.32 ÎźM vemurafenib using an HP D300 Digital Dispenser. Fortyâeight hours after treatment, cells were fixed and analyzed for NGFR expression using immunofluorescence microscopy, as described earlier. To identify candidate compo;tion plates and a Seiko pin transfer robot system. Immediately after, A375 and WM115 cells were then treated with 1 ÎźM vemurafenib and COLO858 cells were treated with 0.32 ÎźM vemurafenib using an HP D300 Digital Dispenser. Fortyâeight hours after treatment, cells were fixed and analyzed for NGFR expression using immunofluorescence microscopy, as described earlier. To identify candidate compo
Read morecells were treated with 5 concentrations of each compound (10-fold dilution from 0.001 to 10 mmol/L in duplicates) using the HP D300 digital dispenser (Tecan). Cell viability was measured 48 hours after drug treatment by colorimetric MTS assay following the supplier's recommendations (Promega). Each experiment was repeated at least twice for each cell line, and results were normalized on untreated cells. Curve fitting of doseâresponse data was performed using GraphPad Prism 6 Software, and the two following classical parameters representative of drug sensitivity were derived: (i) the GI50 corresponding to the concentration of drug that inhibits 50% of cell viability and (ii) the AUC corresponding to the area under the doseâresponse curve that provides an overall measure of cumulative response. When the GI50 was not reached, the values were set to the highest concentration tested (10 mmol/L)
Read moreAssays were performed by seeding 384-well plates with 10,000 cells per well in MEMalpha containing 10% FBS and dosing compounds at indicated concentration using the HP D300 digital dispenser (HP/Tecan)
Read moreted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard, Palo Alto, CA) in 2-fold dilution increments starting at 10 ÎźM at 2 h prior to infection. The DMSO concentration in each well was normalized to 1% using an;ted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard, Palo Alto, CA) in 2-fold dilution increments starting at 10 ÎźM at 2 h prior to infection. The DMSO concentration in each well was normalized to 1% using an
Read moreIC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on ;obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well
Read moreGrowth rates were determined by measuring the OD 600 approximately every 15 min for at least 80 cycles at 30 °C in TECAN sunrise or GENios plate readers. Drugs were dissolved in DMSO and dispensed to plates using an HP D300 Digital Dispenser (Tecan)
Read morevine serum albumin (BSA), pH 7.4) and 100 ÎźL/well dispensed (Multidrop, Thermofisher) into white 96-well plates (Corning) followed by dispensing of test compounds from 10 mM stock solutions using an HP D300 Digital Dispenser (Hewlett-Packard). The LED-array was then placed on top of the 96-well plate and enzyme inhibitor solutions were exposed to light for 1 h (470 nm, 17 mW/cm2). Light exposure;vine serum albumin (BSA), pH 7.4) and 100 ÎźL/well dispensed (Multidrop, Thermofisher) into white 96-well plates (Corning) followed by dispensing of test compounds from 10 mM stock solutions using an HP D300 Digital Dispenser (Hewlett-Packard). The LED-array was then placed on top of the 96-well plate and enzyme inhibitor solutions were exposed to light for 1 h (470 nm, 17 mW/cm2). Light exposure
Read moreth a ViewLux uHTS Microplate Imager (PerkinElmer). Follow-up assays were performed in black 384-well plates (Greiner) in 20 ¾L of enzyme buffer to which compounds were added in dose-response with an HP D300 digital dispenser (Hewlett-Packard), followed by addition of 20 ¾L of substrate buffer. Plates were incubated at room temperature (25 °C) and read at 0 and 20 minutes with a Spectramax M5 pl;Page 1. Š 201 6 Nature America, Inc. All rights reserved. 452 nature chemical biology | vol 12 | June 2016 | www.nature.com/naturechemicalbiology ARTICLE published online: 25 april 2016 | doi: 10.1038/nchembio.2070 One
Read moreCompounds were dispensed in a black 384-well low-volume microplate (Greiner Bio-One) using a Hewlett Packard HP D300 digital dispenser (Tecan Group Ltd.)
Read moreCompound stock solutions in DMSO were titrated into 384-well plates using an HP D300 digital dispenser
Read moreFor pairwise and triple combinations, compound doses were combined at a fixed ratio of 1:1 and 1:1:1, respectively (17). DMSO concentrations were normalized per well. In addition, vehicle (DMSO) and positive controls (staurosporine) were transferred. All treatments were performed in triplicates. One of the replicates received 50 nL of 2 mmol/L CellEvent Caspase-3/7 Green Detection Reagent (Thermo) using a HP D300 dispenser (Tecan). Cells were treated for 72 to 96 hours. Caspase-3/7 activity was imaged every 24 hours. At the end of the treatment, cells were fixed, and their DNA was stained and imaged (18, 19). High-order experiments combining more than 3 drugs were also setup with fixed ratios but only testing 4 dose points. To validate synergies of MDM2 combinations, matrices were tested to assess effects at different ratios of the single agents
Read moreAt 24 hours post-seeding, 100 Âľl of serum-free medium with or without cetuximab (5 Îźg/ml) was manually added to the cells. All other drugs were added directly on the plate by TECAN D300e digital dispenser (HP). After 72 hoursâ treatment cell viability was assessed by ATP content using CellTiter-Glo Luminescent Assay (Promega). Viability was normalized as a percentage of control untreated cells. Data from growth-inhibition assays were plotted using the nonlinear regression curve fit modelling from GraphPad Prism-5 (GraphPad Software)
Read moreFor research use only. Not for use in diagnostic procedures.
Tecan D300e Digital Dispenser is a product of HP Inc. CA, USA.