Confluent cells (30,000/well) were plated in 96-well clear bottom black-sided plates (Costar; Corning, NY, USA) for priming and activation. Cells were incubated with NucGreen Dead 488 ReadyProbes Reagent (Invitrogen; Waltham, MA, USA) for 20 min. Then, 20× images were collected using a Cytation 1 (BioTek; Winiiski, VT, USA). Cell viability was calculated as the average number of green positive cells in each condition, relative to a RIPA lysis control well (0% viability).
Nucgreen dead 488 readyprobes reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NucGreen Dead 488 ReadyProbes Reagent is a fluorescent dye used for detecting dead cells in cell samples. It binds to DNA in cells with compromised cell membranes, providing a simple and reliable method for identifying non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Cytation 1, by Agilent Technologies (1 mentions)
Lightsheet z 1, by Zeiss (1 mentions)
Seaplaque gtg agarose, by Lonza (1 mentions)
Evos fl auto 2 cell imaging system, by Thermo Fisher Scientific (1 mentions)
Evos fl auto 2 microscope, by Thermo Fisher Scientific (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
2 deoxyglucose d6134, by Merck Group (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Nucblue live reagent, by Thermo Fisher Scientific (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
Ethylene glycol, by Merck Group (1 mentions)
8 protocols using nucgreen dead 488 readyprobes reagent
1
Quantitative Cell Viability Assay
2
Nuclear Staining and Clearing Protocol
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At day 5, cells were washed 3 times with warmed PBS for 5 min, followed by paraformaldehyde fixation ( 3.7% in PBS) for 20 minutes. All wells were then washed with PBS/3% BSA (3×5 min), permeabilized with 0.5% Triton for 20 min, and rinsed again with PBS/ 3% BSA (3x 5 min). To stain nucleus, NucGreen Dead 488 ReadyProbes Reagent were used (Invitrogen R37109, 2 drops/mL , i.e. 2 drops per wells, 4 hours at room temperature). The samples were washed with PBS (1×10 min) and kept protected from light in PBS at 4 • C until image acquisition. Such non-cleared samples were either imaged directly (Control), or further processed with different clearing methods, described below.
3
Transparent Imaging of HH16 Embryos
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To examine the effect of SHH inhibition on the phenotype at HH16, embryos were observed by light sheet microscopy with a Zeiss Lightsheet Z.1 (ZEISS) microscope (Fig. 1D ). Because HH16 embryos are large, the CUBIC method (44 (link)) was used to render them transparent to allow deep-tissue imaging. Embryos were fixed with 4% paraformaldehyde (PFA) (Wako, 162-16065) in phosphate-buffered saline (PBS) (Nippon Gene, 314-90185) for 1 hour and washed twice with PBS before treatment with CUBIC-I solution for 24 hours at 37°C. Then, the embryos were stained with NucGreen Dead 488 ReadyProbes Reagent (Thermo Fisher Scientific, R37109) in CUBIC-I solution for 24 hours at 37°C and washed twice with PBS. For imaging, the embryos were mounted with 0.8% SeaPlaque GTG agarose (Lonza, 50111)/PBS.
4
SARS-CoV-2 Infection Assay in A549 Cells
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Human ACE expressing A549 cells were plated at 1 × 104 cell/well in a 96 well plate. After overnight incubation at 37˚C, cells were infected with SARS-CoV-2 at 0.1 MOI for 1 h and then treated with selected concentration then treated with CAP, LYC, ROT and CBLNA at indicated concentrations. DMSO was used as a control. Cells were then incubated 72 hs. Cells were then stained using NucGreen™ Dead 488 ReadyProbes™ Reagent (ThermoFisher) dead indicator, which stains cells that have lost plasma membrane integrity. Cells nuclei were counterstained using DAPI (ThermoFisher). Cell cultures were imaged using an EVOS FL Auto 2 Cell Imaging System (ThermoFisher). Cell death was quantified by enumerating the number of NucGreen positive cells proportional to total cell nuclei per field of view [43].
5
SARS-CoV-2 Infection Cytotoxicity Assay
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A549 cells were stained using NucGreen Dead 488 ReadyProbes reagent (Thermo Fisher Scientific) dead indicator, which stains cells that have lost plasma membrane integrity. Cell nuclei were counterstained using DAPI (Thermo Fisher Scientific). Cell cultures were imaged 72 h after SARS-CoV-2 infection using an EVOS FL Auto 2 microscope (Thermo Fisher Scientific). Cell death was quantified by enumerating the number of NucGreen-positive cells proportional to total cell DAPI-stained nuclei per field of view (17 (link)). Image analysis was performed using ImageJ. Additionally, at 72 h postinfection, extracellular lactate dehydrogenase (LDH) activity was assessed using the CytoTox 96 nonradioactive cytotoxicity assay (Promega) according to the manufacturer’s instructions.
6
Metabolic Intervention and Cell Death
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Cells were treated with 15 mM 2-deoxyglucose (D6134, Sigma Aldrich, MA, USA), 750 μM heptanoate (C7; 75190; Sigma Aldrich, MA, USA). Cell death over a minimum of 72 h was determined using the NucGreen Dead 488 ReadyProbes Reagent (Thermo Fisher Scientific, MA, USA) as per the manufacture's protocol and observed using the IncuCyte S3 (Essen BioScience Inc., MI, USA).
7
Monitoring Cell Viability with Confocal Microscopy
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Cells were seeded into a 96-well plate. After 24 h, cells were stained for 15 min with NucBlue Live ReadyProbes Reagent (1 drop/ml media) (ThermoFisher) in Hank’s balanced salt solution (HBSS) (Life Technologies). Cells were washed once with HBSS and treated with 100 μl of potassium bromate (KBrO3) (Millipore) at concentrations of 0, 1, 10, 100, and 200 mM in HBSS with NucGreen Dead 488 ReadyProbes Reagent (1 drop/ml media) (ThermoFisher).
The 96-well plate was imaged immediately over the course of 24 h every 20 min at 37°C using a Nikon TiEclipse inverted A1 confocal microscope equipped with a 20× objective (NA = 0.75) and driven by NIS Elements AR 4.30.02v 64-bit acquisition software (Nikon). Cells were imaged simultaneously in the fluorescein isothiocyanate (FITC) (NucGreen) and 4′6-diamidino-2-phenylindole (DAPI) (NucBlue) channels. A cell was defined as undergoing cell death when 50% or more of the nucleus, as defined by NucBlue-positive pixels, was overlapped by NucGreen-positive pixels. Cell death was multiplied by 100% and subtracted from 100 to calculate % Viability. Images were analyzed using Python.
The 96-well plate was imaged immediately over the course of 24 h every 20 min at 37°C using a Nikon TiEclipse inverted A1 confocal microscope equipped with a 20× objective (NA = 0.75) and driven by NIS Elements AR 4.30.02v 64-bit acquisition software (Nikon). Cells were imaged simultaneously in the fluorescein isothiocyanate (FITC) (NucGreen) and 4′6-diamidino-2-phenylindole (DAPI) (NucBlue) channels. A cell was defined as undergoing cell death when 50% or more of the nucleus, as defined by NucBlue-positive pixels, was overlapped by NucGreen-positive pixels. Cell death was multiplied by 100% and subtracted from 100 to calculate % Viability. Images were analyzed using Python.
8
Synthesis and Characterization of Multimodal Nanoparticles
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Rhodium(III)
chloride hydrate (RhCl3·xH2O, Rh 38.5%–45.5%), Ruthenium(III)
chloride hydrate (RuCl3·xH2O, Ru 38%–40%), Ethylene glycol (EG, >99%), Poly(vinyl-pyrrolidone)
(PVP, 55 kDa), Ammonium heptamolybdate (AHM, (NH4)6Mo7O24·4H2O), (3-Aminopropyl)triethoxysilane
(APTES, H2N(CH2)3–Si(OC2H5)3), Cy5.5 Mono NHS Ester (Cy5.5-NHS),
Triethylamine (TEA, (C2H5)3N, ≥99%),
Dimethyl sulfoxide (DMSO, (CH3)2SO), Hydrochloric
acid (HCl, 37%), Tetraethyl orthosilicate (TEOS, Si(OC2H5)4, ≥ 99%), Ethanolamine (EA, NH2CH2CH2OH, ≥99%), Dulbecco’s
modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), and Murine
macrophages (RAW 264.7, 91062702-1VL) were all purchased from Sigma-Aldrich.
Ethanol (EtOH, CH3CH2OH, 99.7%) was bought from
Solveco. All fluorescent probes, NucGreen Dead 488 ReadyProbes Reagent
(SYTOX Green), 4′,6-diamidino-2-phenylindole (DAPI), NucBlue
Live reagent (Hoechst 33342 dye), LysoTracker Green DND-26, Alexa
Fluor 555 Phalloidin, and Alexa Fluor 488 Phalloidin were all purchased
from ThermoFisher.
chloride hydrate (RhCl3·xH2O, Rh 38.5%–45.5%), Ruthenium(III)
chloride hydrate (RuCl3·xH2O, Ru 38%–40%), Ethylene glycol (EG, >99%), Poly(vinyl-pyrrolidone)
(PVP, 55 kDa), Ammonium heptamolybdate (AHM, (NH4)6Mo7O24·4H2O), (3-Aminopropyl)triethoxysilane
(APTES, H2N(CH2)3–Si(OC2H5)3), Cy5.5 Mono NHS Ester (Cy5.5-NHS),
Triethylamine (TEA, (C2H5)3N, ≥99%),
Dimethyl sulfoxide (DMSO, (CH3)2SO), Hydrochloric
acid (HCl, 37%), Tetraethyl orthosilicate (TEOS, Si(OC2H5)4, ≥ 99%), Ethanolamine (EA, NH2CH2CH2OH, ≥99%), Dulbecco’s
modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), and Murine
macrophages (RAW 264.7, 91062702-1VL) were all purchased from Sigma-Aldrich.
Ethanol (EtOH, CH3CH2OH, 99.7%) was bought from
Solveco. All fluorescent probes, NucGreen Dead 488 ReadyProbes Reagent
(SYTOX Green), 4′,6-diamidino-2-phenylindole (DAPI), NucBlue
Live reagent (Hoechst 33342 dye), LysoTracker Green DND-26, Alexa
Fluor 555 Phalloidin, and Alexa Fluor 488 Phalloidin were all purchased
from ThermoFisher.
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