For cell viability (cytotoxicity), the cells were seeded at a concentration of 2.5 × 105 cells/well on transwell 24-well plates. Then, the cells were exposed to smoke from one t-cig or e-cig using the ISO 3308:2015 puff topography protocol. The t-cig smoke or e-cig aerosol was diluted to maximum 1:100 and was added directly to the cell culture air–liquid interface. The viability was determined through a colorimetric MTT staining assay according to the manufacturer’s protocol (MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, Steinheim, Germany), as previously described [28 (link)]. The MTT measures the activity of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which is purple in color. The viability measured by the MTT assay was read with an absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a test wavelength of 595 nm with a reference wavelength of 690 nm. The optical density was calculated as the difference between the reference wavelength and the test wavelength. In total, the three independent experiments were conducted in triplicates.
Absorbance microplate reader
Manufactured by Molecular Devices
Sourced in United States
The Absorbance Microplate Reader is a laboratory instrument designed to measure the absorbance of light in multi-well microplates. It quantifies the amount of light absorbed by samples in each well of the microplate, which can be used to determine the concentration of specific analytes or assess the performance of biological or biochemical assays.
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Lab products found in correlation
Ldh cytotoxicity assay kit, by Cell Biolabs (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Maxisorp microtiter plate, by Thermo Fisher Scientific (2 mentions)
Odyssey near infrared imager, by LI COR (2 mentions)
Cck 8 method, by Enzo Life Sciences (2 mentions)
Mtt cell proliferation assay, by Cell Biolabs (2 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Transam nf κb p65 assay kit, by Active Motif (1 mentions)
Transam nf κb p65 transcription factor assay kit, by Active Motif (1 mentions)
Nuclear extraction kit, by Active Motif (1 mentions)
Cck 8 assay kit, by Meilun (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Opaque 96 well plate, by Merck Group (1 mentions)
N9037, by Merck Group (1 mentions)
Fmk001, by R&D Systems (1 mentions)
Human zonulin elisa kit, by Elabscience (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Dcfh da, by Thermo Fisher Scientific (1 mentions)
29 protocols using absorbance microplate reader
1
Cell Viability Assay for Tobacco and E-Cigarette Exposure
2
LDH Cytotoxicity Assay Protocol
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LDH release assay was performed following the manufacturer’s protocol (LDH Cytotoxicity Assay Kit; Cell Biolabs, CA, USA). Briefly, at 24 h post UVA irradiation, by treating with 15 μL triton X-100 solution for 10 min, these wells were set as a positive control to release all the LDH in the cells. A total of 90 μL of cell culture medium and 10 μL LDH substrate were transferred and mixed into a 96-well plate. The mixture was incubated at 37 °C and 5% CO2 in the dark until the OD 450 values of positive control wells no longer increased. The 96-well plate was read at 450 nm wavelength using an absorbance microplate reader (Molecular Devices, CA, USA). The levels of LDH release were standardized with the positive control as 100% cell death.
3
Sulforhodamine B Assay for Cell Viability
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Cell viability was evaluated using a sulforhodamine B (SRB) assay. Cells were incubated in 96-well plates. Then, the cells were fixed with a trichloroacetic acid solution for 1 h. The supernatant was removed, and the plates were washed five times and air dried. SRB (Sigma-Aldrich Corp.) was added to each of the wells for 1 h. After staining, the residual dye was removed, and the wells were washed five times with 1% acetic acid. Tris buffer (20 mM) was added, and then the absorbance of the solution was measured on an absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 562 nm. The absorbance of untreated cells was used as the reference to calculate 100% cell viability.
4
Quantifying Nuclear NF-κB Activation
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Nuclear protein was extracted from the kidney with a nuclear extract kit (Active Motif, Carlsbad, CA, USA) based on the manufacturer’s instructions. The binding activities of free NF-κB p65 in nuclear extracts were determined with the use of the TransAM NF-κB p65 assay kit (Active Motif) following the manufacturer’s protocol. The plate was read at 450 nm using an absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA).
5
NF-κB p65 DNA-binding Activity Assay
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HUVECs in 6-well plates (3 × 105/well) were exposed to TP at the indicated concentrations for 1 hour, followed by incubation with ox-LDL (50 µg/mL) for an additional 30 minutes. After incubation, nuclear protein was extracted from the cells using a nuclear extraction kit (Active Motif, Carlsbad, California), as described by the manufacturer. A specific TransAM NF-κB p65 Transcription Factor Assay Kit (Active Motif) was used to quantify the DNA-binding activity of NF-κB p65 following the manufacturer's instructions. Briefly, extracted nuclear proteins were added to each well coated with an unlabeled oligonucleotide containing the consensus binding site for NF-κB (5′-GGGACTTTCC-3′) and incubated for 1 hour. After washing, a primary antibody directed against the NF-κB p65 subunit was applied and incubated for 1 hour. Subsequently, a secondary antibody conjugated to horseradish peroxidase was added and incubated for 1 hour. A colorimetric reaction was initiated by adding a developing solution. After termination with a stop solution, the plate was read at 450 nm with an absorbance microplate reader (Molecular Devices, Downingtown, Pennsylvania). The protein levels of the nuclear extract were assayed using a protein assay kit and used to normalize the DNA-binding activity of NF-κB p65.
6
CCK-8 Assay for Cell Proliferation
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The CCK‐8 assay kit (Meilunbio) was used to detect cell proliferation. sh‐NC and sh‐NR5A2 cells were inoculated at a density of 4000 cells/100 μL/well in a 96‐well plate, and incubated at 37°C for 1–5 days. Following incubation, CCK‐8 reagent was added at a concentration of 10 μL/well, as per the manufacturer's instructions. The cells were incubated at 37°C for 1.5 h, and the absorbance of the solution was measured at 450 nm using an Absorbance Microplate Reader (Molecular Devices). The experiment was repeated three times, independently.
7
Glucose and Pae Cytotoxicity on Vascular Endothelial Cells
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The cytotoxic effects of glucose and Pae on VECs growth were determined through the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. VECs were grown to 80% confluence and then seeded into a 96-well flat-bottom plate and incubated with DMEM supplemented with 20% FBS. Different concentrations of glucose (5.5, 15.5, 25.5, 35.5, and 45.5 mM) at multiple time points (0, 12, 24, 48, and 72 h) were used. Furthermore, to investigate the effects of Pae on VECs, cells were pretreated with different concentrations of Pae (7.5, 15, 30, 60, and 120 μM) for different time points (0.5, 6, 12, and 24 h) before being stimulated by glucose at a suitable concentration (35.5 mM) for 48 h. Cells were incubated with glucose at 5.5 mM, which is indicated as the normal glucose (NG) group. After treatment, cells were incubated with MTT (20 μL/well) (Sigma Chemical, USA) for 4 h at 37°C. The medium was then removed and DMSO (200 μL/well) was added to solubilize the precipitate. The absorbance then was measured at 490 nm on an absorbance microplate reader (Molecular Devices, USA).
8
ELISA and Immunoblot Analysis of AnAPN1
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For ELISAs, 96-well Maxisorp microtiter plates (Nunc, Fisher Scientific) were coated with the NT135aaAnAPN1 or An. gambiae midgut lysate as previously described8 (link). The optical density (O.D. 450 nm) and reciprocal serum dilutions were measured on a Molecular Devices absorbance microplate reader. End point titers were defined as the highest reciprocal serum dilution giving an O.D. reading greater than that of pre-immune serum plus 3 standard deviations. For immunoblots, NT135aaAnAPN1 or the near full-length S2-expressed AnAPN1 were resolved on a 4-20% Tris-Glycine SDS-PAGE gel and transferred to nitrocellulose membrane. The blot was incubated with 4H5B7 mAb (30 μg/mL) overnight at 4°C and then detected with goat anti-mouse IgG 680LT Ab (1:50,000) for 1 hour at room temp using the Li-Cor Odyssey Near-infrared imager (Li-Cor). Peptide epitope-mapping ELISAs were performed as described8 (link).
9
Assessing Intracellular GSH Levels
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To determine the critical targets of GX, we assessed the changes in the intracellular GSH level. HAECs were treated with GX (LD90). Untreated cells were used as the negative control group. Using a GSH detection kit, GSH was measured immediately after treatment.
The biochemical principle of this protocol is that dithionitrobenzoic acid reacts with sulfhydryl compounds resulting in a yellowish coloration of the compounds, a process which is catalyzed by GSH. Briefly, HAECs (1 × 106) were suspended and lysed by ultrasound after treatment with GX (LD90, 0.12 mM, for 8 days) or BSO. The samples were mixed with GSH (1 mM) and reagent 1 and incubated at 37 °C for 5 min. Following this, a reaction mixture of reagents 2–5 was added to the samples and incubated at 37 °C for 15 min. The GSH levels were measured using an absorbance microplate reader (Molecular Devices, San Jose, CA, USA) at 420 nm.
The biochemical principle of this protocol is that dithionitrobenzoic acid reacts with sulfhydryl compounds resulting in a yellowish coloration of the compounds, a process which is catalyzed by GSH. Briefly, HAECs (1 × 106) were suspended and lysed by ultrasound after treatment with GX (LD90, 0.12 mM, for 8 days) or BSO. The samples were mixed with GSH (1 mM) and reagent 1 and incubated at 37 °C for 5 min. Following this, a reaction mixture of reagents 2–5 was added to the samples and incubated at 37 °C for 15 min. The GSH levels were measured using an absorbance microplate reader (Molecular Devices, San Jose, CA, USA) at 420 nm.
10
Luciferase-based gene delivery assessment
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