The viability of KUP5, Hepa 1-6, J774A.1, and RAW 264.7 cells, as well as BMDMs, primary Kupffer cells, and primary hepatocytes were assessed by the MTS assay. Cells were exposed to the nanoparticles at the indicated concentrations for 24 h in a 96-well plate, followed by removal of the medium and replacement with 120 μL of complete cell culture media containing 16.7% of a MTS stock solution for 1 h at 37 °C. The plates were centrifuged at 2000g for 10 min in an Eppendorf 5430 microcentrifuge with microplate rotor to collect the cell debris and nanoparticles. Subsequently, 100 μL supernatant was removed from each well and transferred into a new 96-well plate. The absorbance was read at 490 nm on a SpectraMax M5e microplate reader (Molecular Devices, Sunnyvale, CA). Cytotoxic damage to the cell membrane in wild type and siRNA-transfected KUP5 cells was also assessed by using the CytoTox 96 Non-Radioactive Cytotoxicity Assay to measure LDH release. Following exposure to nanoparticles at 200 μg/mL in a 96-well plate, 50 μL of each cell supernatant was mixed with 50 μL of reconstituted Substrate Mix, and incubated at room temperature for 30 min. Next, 50 μL of Stop Solution was added to each well and the absorbance was read at 490 nm on a SpectraMax M5e microplate reader (Molecular Devices, Sunnyvale, CA).
Spectramax m5e
Manufactured by Molecular Devices
Sourced in United States, Japan, China
The SpectraMax M5e is a multi-mode microplate reader that can measure various types of absorbance, fluorescence, and luminescence assays. It features high-performance optics and a sensitive detector to enable accurate and reliable measurements across a wide range of applications.
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Lab products found in correlation
Fitc dextran, by Merck Group (3 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (3 mentions)
Celltiter blue assay kit, by Promega (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (2 mentions)
Jem 2100f, by JEOL (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Ip one gq kit, by PerkinElmer (2 mentions)
Superblock buffer, by Thermo Fisher Scientific (2 mentions)
High binding microplate, by Greiner (2 mentions)
Baf807, by R&D Systems (2 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (2 mentions)
96 well plate, by Corning (2 mentions)
48 well cell culture plate, by Corning (2 mentions)
Black 96 well microtiter plate, by Corning (2 mentions)
Ab211091, by Abcam (2 mentions)
Dual luciferase assay kit, by Hanbio Biotechnology (2 mentions)
262 protocols using spectramax m5e
1
Cell Viability Assays for Nanoparticle Toxicity
2
Quantifying Nuclear NF-κB Activation
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The separation of nuclear extracts from cytoplasmic fractions was performed using a Nuclear/Cytosol Extraction kit (BioVision,), Nuclear Extraction kit (Cayman Chemical Company), or NE-PER Nuclear Protein Extraction kit (Thermo Fisher Scientific). Efficient cytoplasmic and nuclear fractionation was confirmed by Western blotting analysis using anti-GAPDH antibody (Cell Signaling Technology) for cytoplasmic fraction and anti-HDAC1 antibody (Santa Cruz Biotechnology, Inc.) for nuclear fraction. NF-κB p65 activity (nuclear DNA binding) was measured with NF-κB (p65) Transcription Factor Assay kit (Cayman Chemical Company) according to the manufacturer’s protocol. ∼1 µg of nuclear proteins were analyzed for each condition. Results were obtained with Spectra Max M5e (Molecular Devices) plate reader (absorbance at 450 nm). The same pools of BMDM were used to measure cytokine levels in parallel to confirm the phenotype, analyzing TNF and IL-1β cytokines with ELISA kits obtained from Thermo Fisher Scientific. Measurements were performed with a Spectra Max M5e (Molecular Devices) plate reader at 450 and 550 nm.
3
Fluorometric Microplate Assay Protocol
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Ethyl-3,4-dephostatin and 3-OMFP were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO and sterile water, respectively. Polydimethylsiloxane (PDMS, Sylgard 184 silicone elastomer kit) was purchased from Dow Corning (Midland, MI, USA), mineral oil was purchased from Sigma-Aldrich, and ABIL EM 90 surfactant was purchased from Evonik Industries (Essen, Germany). A disposable 1.0 mm diameter biopsy punch was purchased from Integra Lifesciences (Princeton, NJ, USA). Glass slides (76 × 26 mm) with a thickness of 1 mm were obtained from Paul Marienfeld (Lauda-Königshofen, Germany). Fluorometric measurements in a microplate were performed using a SpectraMax M5e multi-mode microplate reader from Molecular Devices (San Jose, CA, USA).
4
Cell Viability Assays for Drug Screening
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Two assays were performed. For the Trypan blue exclusion test of cell viability, cells (5 × 105 cells/well) were seeded in 24-well plates. After incubation for the indicated time periods, cells were trypsinized. The suspended cells were mixed with Trypan blue (Sigma) and the number of cells with a clear cytoplasm was counted. For the alamarBlue assay, cells (2500 cells/well) were seeded in 96-well plates and incubated as described for the indicated times. The alamarBlue Cell Viability Regent (Invitrogen, Carlsbad, CA, USA) was added to the cells for 1 h, and immunofluorescence was measured using a SpectraMax M5e (Molecular Devices). Alternatively, cells (2.5 × 104 cells/well) were incubated in 6-well plates for 6 days, and then stained with May-Grunwald Giemsa stain (Muto Pure Chemicals) and analyzed by microscopy. The medium with or without CHK1i (PF-477736) was changed by every 3 days.
5
Thermostability Assay of Aldolase Enzymes
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SEC-purified nanomaterials, as well as the trimeric aldolases I53-40T3 and 4e38 were diluted into “40 buffer” lacking CHAPS such that the total aldolase concentration was 10 μM; 100 μL aliquots of these samples were subjected to a range of incubation temperatures for 1 h. After incubation, samples were centrifuged at 5000 × g for 1 h and then diluted to 0.1 μM. Aldolase activity was measured via an LDH-coupled colorimetric assay. A master mix containing 0.1 mg/mL L-LDH (Sigma), 0.1 mM NADH (Sigma), 1 mM 2-keto-3-deoxy6-phosphogluconic acid (KDPG, Sigma) was prepared and 90 μL aliquots were added to a Corning UV transparent half-area 96-well plate. Ten microliters of diluted aldolase sample was added to each well to initiate the coupled reaction, and the consumption of NADH was monitored via A339 nm on a SpectraMax M5e (Molecular Devices). The relative initial reaction velocity over 100–200 s was calculated via regression analysis (Softmax Pro).
6
Tox Promoter-Driven Luciferase Assay
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A 2.3 kb fragment (−2133 to 232) of the Tox promoter was cloned into the pGL3-Basic vector to drive firefly luciferase expression (the Tox-luciferase reporter). HEK293T cells were cultured in a 96-well plate overnight and were co-transfected with the Tox-luciferase reporter and an empty or NFIL3-encoding vector. A pCMV-Renilla-Luciferase reporter was co-transfected into HEK293T cells to serve as an internal control. Luciferase activities were detected using the Dual-Glo Luciferase Assay kit (Promega) and measured with a SpectraMax M5e plate reader (Molecular Devices, Sunnyvale, California). Firefly luciferase activities in each sample were first normalized against Renilla luciferase activities in the same sample and then normalized against that in cells transfected with the empty vector.
7
Analyzing LuxS-Dependent AI-2 Signaling in H. pylori
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WT, luxSOP, and ΔluxS H. pylori cultures were inoculated into 3 ml BB10 from plates. Four hours after inoculation, cultures were diluted back to 5 × 107 cells ml−1 and incubated with shaking at 37°C and 10% CO2. Samples of WT and luxSOP cultures were taken at 4, 8, 12, 24, 36, and 48 h postdilution and analyzed for optical density and CFU levels on plates. Samples of WT, luxSOP, and ΔluxS cells were taken at each time point and spun down at 13,500 rpm for 3 min, and the cell-free supernatant (CFS) was saved and stored at −20°C. The CFS was analyzed for AI-2 using the Vibrio harveyi luminescence assay. Ten microliters of CFS was added to 90 µl of V. harveyi culture (prepared as previously described for the AI-2 binding assay) with duplicate samples in a 96-well plate. Levels of luminescence were measured on a SpectraMax M5e plate reader (Molecular Devices) each hour over the course of 6 h with incubation on a shaker at 30°C between measurements. Levels of relative luminescence units (RLUs) were taken at the time point where there was the largest difference between an exogenous DPD positive control and the ΔluxS negative control.
8
Heparin Binding Affinity Assay
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Recombinant human Furin with His tag (100 μl) at 2.5 μg/ml were coated onto Immuno transparent flat 96 well plate with hydrophilic surface (Thermo, 439454) at RT for 2 h. Wash three times with 1 × PBS, each time 5 min. The plates were blocked with 3% BSA in 1 × PBS for 1 h at RT. After removing the blocking buffer, biotinylated heparin (1000, 500, 250, 125, 62.5, 31.25, 15.625 μg/ml) was added into the plate in 100 μl and incubated at RT for 1 h, the PBS serves as control. Pierce™ High Sensitivity Streptavidin-HRP (50 ng/ml, Thermo, 21132) was added and incubated for 1 h at RT. Wash with 1 × PBS 6 times. TMB (100 μl, Thermo, UJ2859123) was added and incubated at RT for 30 min. The reaction was stopped by adding 2 M H2SO4. The results were read by a spectrometer SpectraMax M5e (Molecular Devices, USA) at 450 nm.
9
Furin Binding Assay in Cells
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Cells (3.0 × 104 cells/well) were seeded into 96-well plates and incubated overnight at 37 ºC with 5% CO2 in a humidified incubator. The cells were washed by 1 × PBS and then fixed in 4% paraformaldehyde at room temperature (RT) for 15 min. After washed by 1 × PBS for 3 times, the cells were blocked in 3% BSA at RT for 90 min. After removing the blocking buffer, human Furin at 2.5 μg/ml or Furin-heparin mixture with Furin (2.5 μg/ml) and heparin (500 μg/ml) pre-mixed on ice for 30 min was added and incubated at RT for 1 h. The blank served as the control. The cells were washed 3 times with 1 × PBS and incubated with anti-His antibody conjugated with HRP (ZENBIO, 618194, 1:2000) for 1 h at RT. After 6 times washing with 1 × PBS, TMB (Thermo, UJ2859123) was added and incubated at RT for 30 min, the color development was stopped by 2 M H2SO4. Absorbance at 450 nm was measured by SpectraMax M5e (Molecular Devices, USA).
10
Carbohydrate-Furin Binding Assay
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Carbohydrates (100 μl) in different concentration (1000, 500, 250, 125, 62.5, 31.25, 15.625 μg/ml) were coated into Immuno transparent flat 96 well plates with hydrophilic surface (Thermo, 439454) at RT for 2 h. Wash three times with 1 × PBS, each time 5 min. The plates were blocked with 3% BSA for 1 h at RT. Human Furin was added into the plates (0.25 μg/well in 100 μl 1 × PBS) after aspirating the blocking buffer and incubated at RT for 1 h. After 3 times washing by PBS, anti-His antibody conjugated with HRP (1:2000) was added and incubated for 1 h at RT. Wash with 1 × PBS 6 times. TMB (Thermo, UJ2859123) was added and incubated at RT for 30 min. The reaction was stopped by adding 2 M H2SO4. The absorbance of the developed substrate was read by a spectrometer SpectraMax M5e (Molecular Devices, USA) at 450 nm.
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