Recombinant PrP protein in 6M guanidine hydrochloride (GdnHCl) solution was diluted in RT-QuIC buffer (20 mM sodium phosphate pH 7.4; 130 mM NaCl; 10 mM EDTA; 0.002% SDS) to a final protein concentration of 0.2 mg/mL (and residual 0.2 M GdnHCl). Reactions were seeded with AF4 fractions containing the same amount of total PrP (based on immunoblotting intensity) in a final volume of 180 μL/well. The aggregation reactions were carried out in 96-well plates (white plate, clear bottom; Costar 3610) sealed with thermal adhesive film (08-408-240; Fisherbrand). The samples were incubated in the presence of 10 mM thioflavin T (ThT) at 42°C with cycles of 1 min shaking (700 rpm double orbital) and 1 min rest. ThT fluorescence measurements (450+/210 nm excitation and 480+/210 nm emission; bottom read) were collected every 60 minutes. There were three technical replicates per experiment.
Costar 3610
Manufactured by Corning
Sourced in United States
The Costar 3610 is a laboratory equipment product manufactured by Corning. It is designed for general laboratory use. The core function of the Costar 3610 is to provide a reliable and consistent platform for various laboratory experiments and procedures.
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12 protocols using costar 3610
1
Reconstituting Prion Protein Aggregation
2
Prion Seeding Activity Measurement
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Recombinant PrP (moPrP) was solubilized in 6 M guanidine hydrochloride (GdnHCl) solution. This was then diluted in real-time quaking-induced conversion (RT-QuIC) buffer (20 mM sodium phosphate pH 7.4; 130 mM NaCl; 10 mM EDTA; 0.002% SDS) to a final protein concentration of 0.2 mg/mL (and residual 0.2 M GdnHCl). Reactions were seeded with 2 μL of 10% solubilized brain homogenate in a final volume of 180 μL/well. The aggregation reactions were carried out in 96-well plates (Costar 3610) sealed with thermal adhesive film (08-408-240; Fisherbrand). We used three different dilutions of terminal-stage (150 DPI) brain homogenates (10-3, 10-5, and 10-6) as positive controls for these reactions and to calibrate sensitivity. NBH-inoculated mice brain homogenate at 10-3 dilution was used as a negative control. 10-3 dilutions were used to compare seeding activity at different timepoints in prion-infected animals. All test samples were incubated in the presence of 10 mM thioflavin T (ThT) at 42 °C with cycles of 1-min shaking (700 rpm double orbital) and 1-min rest. ThT fluorescence measurements (450+/210 nm excitation and 480+/210 nm emission; bottom read) were recorded every 60 min. There were three technical replicates for each sample per experiment.
3
Quantification of Smad1/5 Phosphorylation
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Human FOP fibroblasts (Coriell Institute) were grown to confluence in DMEM supplemented with 10% FBS (MilliporeSigma) and then seeded into 96-well plates (Costar 3610; Corning). The cells were incubated at 37°C and 10% CO2, allowed to attach, then starved overnight in 1% FBS-DMEM. Saracatinib (Biotang Inc.) was dissolved in DMSO (Fisher Scientific), whereas LDN-193189 (Yu Lab) was diluted in 0.1% PBS-BSA. The cells were then preincubated with increasing molar concentrations of saracatinib and LDN-193189 for 15 minutes. Activin A (R&D Systems) was diluted in 0.1% PBS-BSA (MilliporeSigma) to a working concentration of 250 ng/mL. After addition of Activin A, the cells were allowed to incubate for 45 minutes. Fixation of the cells was performed with ice-cold methanol and 0.5% glutaraldehyde (Fisher Scientific) before the addition of primary antibody (Phospho-Smad1/5 (Ser463/465) rabbit mAb 9516; Cell Signaling Technologies) and secondary antibody (Anti-rabbit IgG, HRP-linked antibody; Cell Signaling Technologies). Finally, the cells were developed with BioFX Chemiluminescent Ultra Sensitive HRP Microwell Substrate solution (Surmodics) and read on a Spectra Max L plate reader (Molecular Devices). Softmax Pro software was used, on the Ready to Glow LUC Assay setting, with an integration time at 0.25 seconds and a wavelength of 470 nm.
4
Luciferase Assay for HMGA2 Evaluation
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For a typical luciferase assay, ∼106 HeLa cells were plated per six-well and co-transfected the following day with 1 μg of both HMGA2 expression and reporter vector using Lipofectamine 2000 (Invitrogen/Life Technologies). About 40 000 cells were transferred the next day per 96-well on a white plate with clear-bottom (COSTAR 3610, Corning) in 150 μl medium. After 24 h recovery, cells were treated for additional 24 h with compounds (dissolved in DMSO), as indicated in the text. Luciferase readings were performed with ‘Renilla Glo’ E2710 kit (Promega) following the manufacturer's instruction on a TECAN Infinite M200 Pro bioluminescence reader.
5
Cytotoxicity and Cytokine Secretion Assay
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Cytotoxicity was measured using CytoTox-Glo (Promega) following the manufacturer's protocol with minor modifications. Primary T cells expanded from healthy donor PBMC as described above were used as effector cells and IGROV-1, SKOV-3, or JeKo-1 cells were used as target cells. The cells were incubated at an effector-to-target (E:T) ratio of 10:1 in X-VIVO 20 Medium (Lonza) with 5% (v/v) off-the-clot human serum AB (Innovative Research). The target cells (2 × 104) were first incubated with the biAbs prior to adding the effector cells (2 × 105) in a final volume of 100 μL/well in a 96-well tissue culture plate. The plates were incubated for 16 h at 37 °C with biAb concentrations ranging from 0.08 to 500 nM. After centrifugation, 50 μL of the supernatant was transferred into a 96-well clear bottom white walled plate (Costar 3610; Corning) containing 25 μL/well CytoTox-Glo. After 15 min at room temperature, the plate was read using a SpectraMax M5 instrument with SoftMax Pro software. The same supernatants (diluted 10-fold) used for the CytoTox-Glo assay were also used to determine IFN-γ, IL-2, and TNF-α secretion with Human IFN-γ, IL-2, or TNF-α ELISA MAX™ Deluxe kits (BioLegend), respectively, following the manufacturer's protocols.
6
Cell Viability Assay of Hepatocarcinoma Cells
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HepG2 hepatocarcinoma cells were
seeded in DMEM supplemented with 10% FBS at 25000 cells per well in
tissue culture treated 96-well plates (Costar 3610; Corning). The
cells were incubated for 2 h (37 °C and 5% CO2) and
allowed to settle and attach. Compounds of interest or DMSO were diluted
in DMEM and added at final compound concentrations of 1, 10, and 100
μM. Cells were incubated for 4 and 24 h, after which the media
was discarded. Cells were lysed by adding 30 μL of passive lysis
buffer (Promega) and shaken at RT for 15 min. Cell viability was determined
by quantifying the ATP present in each well by adding 10 μL
of Cell Titer Glo (Promega) per well and measuring the light output
Spectramax L luminometer (Molecular Devices) with an integration time
of one second per well. Data was normalized to 100% viability for
cells receiving only DMSO without any concurrent compound.
seeded in DMEM supplemented with 10% FBS at 25000 cells per well in
tissue culture treated 96-well plates (Costar 3610; Corning). The
cells were incubated for 2 h (37 °C and 5% CO2) and
allowed to settle and attach. Compounds of interest or DMSO were diluted
in DMEM and added at final compound concentrations of 1, 10, and 100
μM. Cells were incubated for 4 and 24 h, after which the media
was discarded. Cells were lysed by adding 30 μL of passive lysis
buffer (Promega) and shaken at RT for 15 min. Cell viability was determined
by quantifying the ATP present in each well by adding 10 μL
of Cell Titer Glo (Promega) per well and measuring the light output
Spectramax L luminometer (Molecular Devices) with an integration time
of one second per well. Data was normalized to 100% viability for
cells receiving only DMSO without any concurrent compound.
7
Real-Time Cellular Necrosis and Apoptosis Assay
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Cellular necrosis and apoptosis were measured by a RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kit (Promega, Madison, WI, USA) according to manufacturer’s instruction. Briefly, 1 × 104 SNB-19 cells/well were seeded into a 96-well plate (Costar 3610, Corning, NY) and cultured at 37 °C/5% CO2 overnight. The cells were transduced with adenovirus, and 2x detection reagent (which included Annexin V NanoBiT® substrate, CaCl2, Necrosis Detection Reagent, Annexin V-SmBiT and Annexin V-LgBiT) was added into each tested well of 96-well plate. The plates were incubated at 37 °C/5%, followed by measurements of luminance (RLU) for apoptosis, and fluorescence (RFU, 485 nmEx/520–30 nmEm) for necrosis at the indicated time intervals post infection using a SYNERGY-H1 microplate reader.
8
Bioluminescent Zebrafish Infection Assay
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The Tg (pck1:luc1) zebrafish line obtained from the laboratory of Dr. Stainier (Gut et al., 2013 (link)) was out-crossed with the lepbibl55 mutant (8 bp deletion) to obtain a heterozygous lepbibl55/+Tg (pck1:luc1) line. Subsequently, the heterozygous adult zebrafish were in-crossed, raised and genotyped for lepb+Tg (pck1:luc1) and lepbibl55Tg (pck1:luc1) zebrafish lines. The embryos of the two lines were injected into the yolk with 300 colony-forming unit (CFU) M. marinum strain M labelled with mWasabi plasmid pTEC15 vector22 or mock injected with 2% pvp40 at 4 to 6 hpf. Zebrafish larvae at 5 dpf were washed and transferred (one larva/well) into a 96-well flat clear bottom white polystyrene TC-treated microplate (Corning Incorporated, Costar 3610). 100 μL egg water and 100 μL steady-glo (Promega) were added to the wells followed by incubation of 30 min at room temperature to generate a bioluminescence signal. The luminescence signal was then detected by a microplate reader (TECAN, Infinite M1000).
9
Organoid Gemcitabine Dose-Response Assay
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Organoids were mechanically dissociated before being resuspended in 2% matrigel/growth media seeded (500 cells/well) and dispensed in 96-well plates (Costar #3610; Corning, NY, USA). Cells were incubated at 37 °C/5% CO2 overnight and then exposed to gemcitabine (Calbiochem #504594, Merck-Millipore, Darmstadt, Germany) using a 11-point dose-response curve with a 1:3 dilution series from 10 μM to 0.2 nM. After 72 h, relative viability was measure by CellTiter-Glo 3D cell viability assay (#G9683; Promega, MA, USA). Dose-response curves were performed in technical and biological (different passage) triplicates. Drug concentrations (molar units) were converted to log-scale and IC50 and IC90 concentrations were calculated by using GraphPad Prism (San Diego, CA, USA).
10
Luminescent Viability Assay for Cell Proliferation
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A luminescent cell viability assay (CellTiter-Glo, Promega, USA) was assessed to determine the cell proliferation rate based on quantitation of the ATP, an indicator of metabolically active cells. Three thousand cells were plated in each well of a 96-well white plate with clear bottom (Costar 3610, Corning Incorporated, USA). Each cell condition was plated in 6 or 10 wells. Cells were allowed to adhere for 24 h, before counting. The homogeneous assay procedure involves addition of 100 μL of the CellTiter-Glo reagent directly to the cells cultured in 100 μL of their corresponding complete medium, before measuring the relative luminescence using a multiplate reader (POLAR Star Omega BMG LABTECH). The experiments were conducted over a week, with counting performed every 24 h for HCT116 and HEK293, and at 96 h for the lentivirus transduction.
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