The right lobes of lungs collected after 21 D were homogenized for 3 minutes in PBS at 50 mg tissue per ml, using a tissue homogenizer (Fisher Scientific, USA). 100 µL of each sample was transferred into low protein-binding microtubes, followed by adding 400 µL acetic acid (0.5 M) and pepsin (0.1 mg/mL in 0.5 M acetic acid) for overnight incubation at 4 °C. Cellular debris was pelleted by 10 min centrifugation at 3500rpm. 30 µL supernatant from each sample was analyzed for total protein content, using the Quick Start™ Bradford Protein Assay (BIO-RAD, USA) according to the manufacturer’s instructions. The remaining 470 µL supernatant was used for collagen extraction by the Sircol™ Soluble Collagen Assay kit (Biocolor Ltd., Carrickfergus, UK) according to the manufacturer’s instructions. Similar prepared collagen standards (0–50 µg) were run in parallel. After collecting the collagen pellets and adding alkali reagent, absorbance was read at 555 nm in a plate reader (SpectraMax M5 microplate, Molecular Devices Corp., Sunnyvale, CA). Data were normalized by total protein content and expressed as µg of soluble collagen per mg of lung protein.
Spectramax m5 microplate
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M5 microplate reader is a versatile instrument designed for a wide range of photometric and fluorometric assays. It features a monochromator-based optical system that enables the measurement of absorbance, fluorescence, and luminescence in microplates. The instrument supports a variety of microplate formats and can accommodate sample volumes ranging from 2 to 800 μL.
Automatically generated - may contain errors
Lab products found in correlation
Tissue homogenizer, by Thermo Fisher Scientific (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
Sircol soluble collagen assay kit, by Biocolor (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Menadione solution, by Merck Group (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide, by Merck Group (1 mentions)
11 protocols using spectramax m5 microplate
1
Quantification of Lung Collagen Content
2
Icariin Effects on Cancer Cell Viability
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MTT assays were used to assess the viability of icariin‐treated cancer cells. Cells were seeded into a 96‐well plate and treated with different concentrations of icariin for the indicated times. Then, 20 μL of MTT solution (5 mg/mL in cell culture medium) was added into each well, and the cells were incubated for another 4 h at 37°C. Then, 150 μL DMSO was added to dissolve the formazan precipitates, and the color absorbance was measured at 570 nm using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices). All experiments were conducted in triplicate.
3
Evaluating Cell Proliferation Assays
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Cell proliferation was assessed using MTT and colony formation assays. For the MTT assay, A549, H1299, H460 and 16HBE cells were seeded at a density of 1×104 cells/well in 96-well plates for 0, 24, 48 and 72 h. MTT (20 µl, 5 mg/ml) was then added to each well at the indicated times and incubated for 4 h at 37°C. Subsequently, MTT solution was removed and replaced with 150 µl DMSO. The cell viability was measured using a SpectraMax M5 microplate reader (Molecular Devices) at 570 nm. For the colony formation assay, A549, H1299, H460 and 16HBE cells (1×103 cells/well) were seeded in 6-well plates and cultured for 14 days at 37°C in a humidified incubator with 5% CO2. Following two washes with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.5% crystal violet for 4 h at room temperature. Cell colonies were counted and photographed using a light microscope (magnification, ×40).
4
Preparation and Characterization of AGEs
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AGEs were prepared through the reaction of fructose and BSA in accordance with the method reported by Zeng et al. [44] (link). with minor modifications. First, 1.5 g of BSA was dissolved in 50 mL of phosphate-buffered saline (PBS; pH 7.4, 0.2 M), and then the BSA solution (30 mg/mL) was incubated with or without 500 mM fructose for 60 days at 37 °C. After incubation, the reaction mixture was placed in a 10 kD dialysis bag and dialyzed in PBS (pH 7.4, 0.2 M) at 4 °C for 24 h. Thereafter, the AGEs were lyophilized and stored at −20 °C until use. Since the AGEs can exhibit self-fluorescence, the fluorescence value of the reaction products was measured for characterizing AGEs [45] (link). In the current study, the fluorescence value was measured at 370 nm excitation and 440 nm emission wavelengths by using a SpectraMax M5 microplate (Molecular Device, Sunnyvale, CA, USA). The AGEs-specific fluorescence value of the AGEs solution (equivalent to 1.0 mg/mL of BSA, 4582.07 ± 19.85) was significantly higher than that of the control group (1.0 mg/mL of BSA, 86.05 ± 4.81; p < 0.05), indicating that the AGEs were prepared well (Fig. S1).
5
MTT Assay for SKLB-163 Cytotoxicity
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The cell viability of SKLB-163-treated cancer cells was determined by the MTT assay. Briefly, cells (4–5 × 103) were seeded in 96-well plates and cultured for 24 h, followed by SKLB-163 treatment for 48 h. A volume of 10 μl of 10 mg/ml MTT was added per well and incubated for another 3 h at 37 °C, then the supernatant fluid was removed and DMSO was added at 150 μl/well for 15–20 min. The light absorptions (OD) were measured at 570 nm with SpectraMAX M5 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The effect of SKLB-163 on tumor cells viability was expressed by IC50 of each cell lines.
6
Preparation and Characterization of AGEs
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AGEs were prepared through the reaction of fructose and BSA in accordance with the methods reported by Zeng with minor modifications [14 (link)]. First, 1.5 g of BSA was dissolved in 50 mL of phosphate-buffered saline (PBS; pH 7.4, 0.2 M), and then the BSA solution (30 mg/mL) was incubated with or without 500 mM fructose for 60 days at 37 °C. After incubation, the reaction mixture was placed in a 10 kD dialysis bag and dialyzed in PBS (pH 7.4, 0.2 M) at 4 °C for 24 h. Thereafter, the AGEs were lyophilized and stored at −20 °C until use. Considering that AGEs exhibit self-fluorescence, the fluorescence value of their reaction mixture was measured to determine AGE production [14 (link)]. In the current study, the fluorescence value was measured at 370 nm excitation and 440 nm emission wavelengths by using a SpectraMax M5 microplate (Molecular Device, Sunnyvale, CA, USA) (Figure S1 ). The fluorescence value of the AGE solution (equivalent to 1.0 mg/mL of BSA, 5572.23 ± 19.95) was significantly higher than that of the control group (1.0 mg/mL of BSA, 77.77 ± 2.99; p < 0.05), indicating that the AGEs were prepared well.
7
Cytotoxicity of EC Aerosols in NHOK Cells
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The cytotoxicity of EC aerosols in NHOK cells was determined by a ATP assay using the ATPliteTM firstep (Perkin-Elmer, Boston, MA) [12 (link)]. After 24 h exposure to EC aerosol-impinged culture medium in a 96-well plate, the culture medium was removed, and cells were washed three times with PBS and incubated with 100 μL of reconstituted ATPlite firstep reagent for 10 min. The luminescence intensity was recorded on a SpectraMax M5 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).
8
Cell Viability Quantification via CCK-8 Assay
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CCK-8 kit (Beyotime, Shanghai, China) was used to measure cell viability. The transfected cells were seeded into a 96-well plate (3000 cells/well) with culture medium refreshed every day. After cell culture for 48 h, the proliferation ability of cells was measured based on following procedure. CCK-8 (10 μL) was added into each well and incubated with cells at 37°C for 2 h. The optical density at 450 nm was measured using SpectraMax M5 microplate (Molecular Devices, San Jose, CA, USA). The cell viability of cells in control group was set as 1, based on which the cell viability in experimental groups was calculated accordingly.
9
BMDM Viability Assay with Fe-NTA
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1×106 BMDM plated in 96 well plates were treated with Fe-NTA (150, 200, 400, 800 µM) or 0.1% saponin for 24 h, washed in PBS, and incubated in medium containing 10% Alamar Blue Cell Viability Reagent (Invitrogen) at 37°C for 4 h protected from light. The fluorescence signal generated by the fluorimetric redox indicator was detected using a SpectraMAX M5 microplate fluorimeter at 560 nm excitation and 590 nm emission, and analyzed using software SoftMAX R _ PRO (Molecular Devices).
10
Quantifying Microbial Metabolism via XTT Assay
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The XTT assay is a colorimetric cell metabolism test, which estimates the microbial active metabolism by reducing the tetrazolium salt to formazan by dehydrogenase enzymes present in the electron transport system of viable cells. The XTT salt (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, Sigma, MO, USA) was prepared in ultra-purified water at the final concentration of 1 mg/mL. The solution was filtered and stored at -80 °C. Menadione solution (Sigma, MO, USA) was prepared in 0.4 mM acetone before the experiment.
At the end of the experimental period, the HA discs were transferred to a falcon tube containing 1 mL of XTT solution (comprised of 158 μL of PBS with 200 mM glucose, 40 μL of XTT and 2 μL of diluted menadione) and the tubes were incubated for 3 h in the dark at 37 °C under anaerobic conditions. After 3 h incubation, 100 μL from each tube was transferred into a 96-well plate and analyzed at 450 nm using a microplate reader (Spectramax ® M5 Microplate, Molecular Devices). Test specimen results were normalized by controls (SH-L-S-) incubated for the same time as treatment groups and expressed as a percentage.
At the end of the experimental period, the HA discs were transferred to a falcon tube containing 1 mL of XTT solution (comprised of 158 μL of PBS with 200 mM glucose, 40 μL of XTT and 2 μL of diluted menadione) and the tubes were incubated for 3 h in the dark at 37 °C under anaerobic conditions. After 3 h incubation, 100 μL from each tube was transferred into a 96-well plate and analyzed at 450 nm using a microplate reader (Spectramax ® M5 Microplate, Molecular Devices). Test specimen results were normalized by controls (SH-L-S-) incubated for the same time as treatment groups and expressed as a percentage.
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