The sensitivity assay was carried out as described here (Toussaint and Conconi, 2006 (link)). In brief, yeast cells were grown to mid-log-phase (OD600 of 0.5–0.7). Cells were collected, washed and resuspended in YPD to a final density of OD 0.8. 5 ul of the cell suspensions were added to 195 ul of YPD with CHX at various concentrations, from 0–10 μg/mL. All samples were prepared in biological triplicates as well as technical duplicates in 96-well polystyrene microplates. The plate was incubated at 30°C with shaking on a microplate scanning spectrophotometer (Biotek). Cell density was monitored every 10 min over 24–48 h at 600nm.
Microplate scanning spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The Microplate scanning spectrophotometer is a laboratory instrument designed to measure the absorbance or optical density of samples in a microplate format. It can perform rapid and accurate absorbance measurements across multiple wavelengths, enabling various applications in biochemistry, cell biology, and analytical chemistry.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
24 well transwell system, by Corning (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by MedChemExpress (1 mentions)
L929 cell line, by American Type Culture Collection (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
7 protocols using microplate scanning spectrophotometer
1
Yeast Sensitivity Assay with Cycloheximide
2
Transwell Invasion Assay for Cell Migration
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The transwell invasion assay was performed as described previously [61 (link)]. Insert wells of 24-well Transwell system (Corning®, NY, USA) were coated with Matrigel (100 μL/well) in 0.7% NaCl (0.01M Tris-HCl, pH 8.0). Cells (7×104/well) in serum-free medium (250 μL) were layered on each insert. AE (0-1000 μg/mL) in medium (600 μL/well) containing 10% FBS was added to wells. At 24 h, inserts were fixed in cold 100% methanol for 20 min, stained with 0.1% crystal violet in 20% methanol for 10 min at room temperature and washed with deionized water. Cells remaining on the upper side of the membranes were removed with cotton swabs and inserts were dried overnight in dark. Stained membranes were photographed in three random non-overlapping fields using a digital photomicroscope (Leica Microsystems, Buffalo Grove, IL, USA).
For quantitative analysis of cell migration, inserts were covered with 300 μL of 10% acetic acid and optical density (OD) of extracted crystal violet was measured at 590 nm using a microplate scanning spectrophotometer (BioTek, Winooski, VT, USA). Cell-free inserts containing only medium were used as OD background controls. Results are presented as average background (OD) - experimental (OD) ± SEM obtained from three independent experiments performed in duplicates.
For quantitative analysis of cell migration, inserts were covered with 300 μL of 10% acetic acid and optical density (OD) of extracted crystal violet was measured at 590 nm using a microplate scanning spectrophotometer (BioTek, Winooski, VT, USA). Cell-free inserts containing only medium were used as OD background controls. Results are presented as average background (OD) - experimental (OD) ± SEM obtained from three independent experiments performed in duplicates.
3
Biocompatibility Evaluation of M13 Phage
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Biocompatibility of M13 bacteriophage (at a concentration of 1012 pfu. mL−1) was examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, USA) assay. Briefly, HUVECs were cultured on surfaces coated with M13 phage, M13 phage-RGD, and gelatin and control plates (no coated surface) at a density of 104 cells per well of 96 well. After 48 hours, MTT solution (20 μL of 5 mg. mL−1 MTT solution) was added to each well, and plates were incubated at 37 °C for 3–4 hours. Then, the medium was discarded, and formazan crystals were dissolved by adding 200 μL of dimethyl sulfoxide (Sigma-Aldrich, USA). The optical density was measured at 570 nm using a microplate scanning spectrophotometer (BioTek, USA).
4
Evaluating Cytotoxicity of Biomaterial Extracts
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The viability of the L-929 cell line (ATCC® Manassas, VA, USA) was assessed using an indirect MTT (3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) colorimetric assay (MedChemExpress, Monmouth Junction, NJ, USA). Extracts of fragmented biomaterials (HGO1, HGO2, BGO1 and BGO2) were prepared by depositing them on inserts for 24 h in a DMEM culture medium. Subsequently, these extracts, along with a positive control (CP, 0.1% Triton/DMEM), were added to cells seeded in 96-well plates at densities of 1 × 104/well, 5 × 103/well and 2.5 × 103/well. The cells were then incubated at 37 °C, 5% CO2 in the DMEM medium for 24, 48, and 72 h, respectively. At the end of the incubation period, the medium was removed and 1 mg/mL MTT (Sigma Aldrich®, USA) was added. The absorbance of formazan was measured at 570 nm using a microplate scanning spectrophotometer (BioTek, Winooski, VT, USA). Cell viability was calculated in comparison to untreated cell culture (negative control, CN) using the formula
where OD sample is the absorbance of the sample at λ = 570 nm (average of 5 replicates) and OD negative control is the absorbance of the negative control at λ = 570 nm (average of 6 replicates).
According to the current ISO 10993-5:2009(E) norm [56 ], a material that does not show cytotoxicity is one for which the cell viability is at least 70%
where OD sample is the absorbance of the sample at λ = 570 nm (average of 5 replicates) and OD negative control is the absorbance of the negative control at λ = 570 nm (average of 6 replicates).
According to the current ISO 10993-5:2009(E) norm [56 ], a material that does not show cytotoxicity is one for which the cell viability is at least 70%
5
Yeast cell growth sensitivity assays
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Sensitivity assays were conducted essentially as described [74 (link)]. Yeast cells were grown to mid-log-phase (OD600 of 0.5–0.7), collected, washed and resuspended in YPD to a final density of OD 0.8. 5 μl of the cell suspension was added to 195 μl of YPD with CHX at various concentrations, from 0–10 μg/mL. All samples were prepared in biological triplicates as well as technical duplicates in 96-well polystyrene microplates. The plate was incubated at 30°C with shaking on a microplate scanning spectrophotometer (Biotek). Cell density was monitored every 10 min over 24–48 h at 600nm. To assay sensitivity to 4NQO, after growing cells to mid-log (OD 0.5–0.7) cultures were treated with and without 5 μg/mL 4QNO for 30 minutes. Cells were collected, washed and adjusted to OD 0.8. Samples were plated and growth monitored as above.
6
Quantifying Cellular Oxidative Stress
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At the 24 th h after X-ray irradiation, the intracellular reactive oxygen species (ROS) levels were assessed by dihydroethidium staining as described previously [24, 27] . In brief, at the end of treatment, the cells were incubated with 10 mM of dihydroethidium for 1 h at 37°C. After washing with PBS, the fluorescence signal was captured under a fluorescence microscope Carl Zeiss Axioplan-2. The mean fluorescence intensity per nucleus was quantified using Image J software. At least 100 nuclear per sample were evaluated at 400× magnification.
Hydrogen peroxide assay MC3T3 cells were cultured in the 24-well plates and immediately exposed to 6 Gy X-rays after CeO 2 nanoparticles treatment. At the 0 and 3 rd h after irradiation, culture medium was collected and medium H 2 O 2 concentrations were measured using Amplex Red Hydrogen Peroxide Assay kit according to the manufacturer's protocols. After incubation with Amplex Red reagents at room temperature for 30 min in the dark, fluorescence intensity was detected at 560 nm excitations and 590 nm emissions using the microplate scanning spectrophotometer (Bio-Tek, Winooski, VT, USA).
Hydrogen peroxide assay MC3T3 cells were cultured in the 24-well plates and immediately exposed to 6 Gy X-rays after CeO 2 nanoparticles treatment. At the 0 and 3 rd h after irradiation, culture medium was collected and medium H 2 O 2 concentrations were measured using Amplex Red Hydrogen Peroxide Assay kit according to the manufacturer's protocols. After incubation with Amplex Red reagents at room temperature for 30 min in the dark, fluorescence intensity was detected at 560 nm excitations and 590 nm emissions using the microplate scanning spectrophotometer (Bio-Tek, Winooski, VT, USA).
7
Cell Viability Assay with MTT
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After X-ray irradiation or H 2 O 2 exposure along with or without CeO 2 nanoparticles treatment, the cell viability was measured with MTT assay kit as described previously [25, 26] . In brief, the MC3T3-E1 cells were incubated with the MTT reagent at 37°C for 4 h, and then the formed formazan crystals was dissolved in 200 μL of dimethyl sulphoxide (DMSO). Optical density (OD) was detected at 490 nm using the microplate scanning spectrophotometer (Bio-Tek, Winooski, VT, USA).
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