A549 cell (100 μL, 5 × 104/mL) were seeded in a 96-well plate, cultured overnight and then treated with different concentrations (200, 400 and 600 μg/mL) of BRBS for 24, 48 and 72 h. Cisplatin (20 μg/mL) was used as a positive control. After centrifugation at 1200 rpm for 7 min, supernatant was discarded and 100 μL of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] (Sigma) solution (0.5 mg/mL in PBS) was added to each well. After 4 h, 150 μL DMSO was added into each well to dissolve the formed formazan crystals. The optical density at 490 nm was measured by a 96-well microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
96 well microplate reader
Manufactured by Bio-Rad
Sourced in United States, Japan
The 96-well microplate reader is a laboratory instrument designed to measure the absorbance, fluorescence, or luminescence of samples in a 96-well microplate format. It provides a standardized and automated method for analyzing multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (12 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (2 mentions)
Mtt reagent, by Merck Group (2 mentions)
96 well plate, by Corning (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Gefitinib, by AstraZeneca (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
Ez cytox cell viability assay kit, by Daeil Lab Service (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mtt assay, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Prl tk, by Promega (1 mentions)
Prl tk, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Mts assay, by Promega (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sirius red, by Merck Group (1 mentions)
62 protocols using 96 well microplate reader
1
Cytotoxicity Assay of BRBS in A549 Cells
2
Antiproliferative Effects of CTPG on Cancer Cells
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The antitumor effects of CTPG on HepG2 and BEL-7404 cells were assessed using MTT
(3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide). HepG2 and
BEL-7404 cells were plated into 96-well plates (5 × 104 cells/well)
and treated with 0, 200, 400, and 600 μg/mL CTPG for 24 and 48 hours,
respectively, after 24 hours of incubation at 37°C. About 35 μg/mL cisplatin
(Yuanye) was used as positive control. Then 100 μL MTT (0.5 mg/mL, diluted by
medium without FBS medium) was added to each well and cultured for 3 hours at
37°C and 5% CO2. After incubation, the plates were centrifuged at
1200 rpm for 7 minutes, the medium was removed and 200 μg/mL DMSO was added to
each well to dissolve the formed formazan crystals. The OD490 values were
measured by a 96-well microplate reader (Bio-Rad Laboratories, CA, USA). To
evaluate the effects of CTPG on splenocytes, the cells were isolated from
C57BL/6 mice and plated into 96-well plates at a density of
1 × 105 cells/well. Splenocytes were treated with 0, 200, 400, and
600 μg/mL for 24, 48, and 72 hours, respectively. The cell viability was
calculated as the followed formula: Cell viability
(%) = (ODtreated/ODuntreated) × 100%.
(3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide). HepG2 and
BEL-7404 cells were plated into 96-well plates (5 × 104 cells/well)
and treated with 0, 200, 400, and 600 μg/mL CTPG for 24 and 48 hours,
respectively, after 24 hours of incubation at 37°C. About 35 μg/mL cisplatin
(Yuanye) was used as positive control. Then 100 μL MTT (0.5 mg/mL, diluted by
medium without FBS medium) was added to each well and cultured for 3 hours at
37°C and 5% CO2. After incubation, the plates were centrifuged at
1200 rpm for 7 minutes, the medium was removed and 200 μg/mL DMSO was added to
each well to dissolve the formed formazan crystals. The OD490 values were
measured by a 96-well microplate reader (Bio-Rad Laboratories, CA, USA). To
evaluate the effects of CTPG on splenocytes, the cells were isolated from
C57BL/6 mice and plated into 96-well plates at a density of
1 × 105 cells/well. Splenocytes were treated with 0, 200, 400, and
600 μg/mL for 24, 48, and 72 hours, respectively. The cell viability was
calculated as the followed formula: Cell viability
(%) = (ODtreated/ODuntreated) × 100%.
3
Antitumor Effects of PFEE-C and PFEE-W
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The antitumor effects of PFEE-C and PFEE-W on H22 and HepG2 cells were assessed by MTT [3-(4,5-dimethyl-2- thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide] (Sigma, MO, USA) assay. H22 and HepG2 cells at the logarithmic growth phase were seeded in 96-well plates at a density of 5 × 103 cells/well and cultured overnight. Cells were treated with PFEE-C and PFEE-W at various concentrations (0, 1.368, 2.736, 5.472, 8.208 μg/ml) of flavonoids or 0.6% DMSO (equal to that in the highest dose of PFEE) for 24, 48 and 72 h, respectively. Cisplatin was used as a positive control. After centrifugation at 1200 rpm for 7 min, supernatant was discarded and 100 μl of MTT solution (0.5 mg/ml in PBS) was added to each well. After incubation for 4 h at 37 °C, the formed formazan crystals were dissolved in 150 μl DMSO. The OD490 values were measured by a 96-well microplate reader (Bio-Rad Laboratories, CA, USA).
To evaluate the effects of PFEE-C and PFEE-W on splenocytes, cells were isolated from C57BL/6 mice and plated into 96-well plates at a density of 1 × 105 cells/well. Splenocytes were treated with PFEE-C and PFEE-W according to the above concentrations for 24 h and 48 h. The relative cell viability was calculated as the followed formula: Cell viability (%) = (ODtreated/ODuntreated) × 100%.
To evaluate the effects of PFEE-C and PFEE-W on splenocytes, cells were isolated from C57BL/6 mice and plated into 96-well plates at a density of 1 × 105 cells/well. Splenocytes were treated with PFEE-C and PFEE-W according to the above concentrations for 24 h and 48 h. The relative cell viability was calculated as the followed formula: Cell viability (%) = (ODtreated/ODuntreated) × 100%.
4
Osteoclast Inhibition by P. coreanum Powder
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Osteoclast precursors were seeded on a 48 well culture plate in the presence of M-CSF (30 ng/mL) and were treated with or without P. coreanum powder (500 μg/mL) for 24 h. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL) was added, and that was followed by 1 h incubation. The absorbance was determined at 570 nm using a 96 well microplate reader (BioRad, Hercules, CA, USA).
5
Quantifying H2S in Trigeminal Ganglion
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H2S level was measured using a previously described method
[26 (link),27 (link)]. Briefly, trigeminal ganglion tissues were homogenized in 250 μl of ice-cold 100 mM potassiumphosphate buffer (pH = 7.4) containing trichloroacetic acid (10% w/v). Zinc acetate (1% w/v, 250 μl) was injected to trap the generated H2S. A solution of N,N-dimethyl-p-phenylenediamine sulfate (20 μM; 133 μl) in 7.2 M HCl and FeCl3 (30 μM; 133 μl) in 1.2 M HCl was added. Absorbance at 670 nm of the resulting mixture (250 μl) was determined after 10 min using a 96-well microplate reader (Bio-Rad). The H2S concentration of each sample was calculated against a calibration curve of NaSH (0–250 μM) and results were expressed as nmol/mg proteins.
[26 (link),27 (link)]. Briefly, trigeminal ganglion tissues were homogenized in 250 μl of ice-cold 100 mM potassiumphosphate buffer (pH = 7.4) containing trichloroacetic acid (10% w/v). Zinc acetate (1% w/v, 250 μl) was injected to trap the generated H2S. A solution of N,N-dimethyl-p-phenylenediamine sulfate (20 μM; 133 μl) in 7.2 M HCl and FeCl3 (30 μM; 133 μl) in 1.2 M HCl was added. Absorbance at 670 nm of the resulting mixture (250 μl) was determined after 10 min using a 96-well microplate reader (Bio-Rad). The H2S concentration of each sample was calculated against a calibration curve of NaSH (0–250 μM) and results were expressed as nmol/mg proteins.
6
Cell Viability Assay with Tetracycline
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786-O and 769-P cells were seededin 96-well plates (1×104 cells/well, 90% density) and exposed to different doses of Tet. Then, 0.5 mg/mL MTT dye solution was added to each well, and the cells were incubated at 37°C for 4 h. Subsequently, the culture medium was discarded, and dimethyl sulfoxide (DMSO) was added to solubilize the precipitate. A 96-well microplate reader (Bio-Rad, Hercules, CA, USA) was used to estimate the absorbance at 490 nm.
7
α-Glucosidase Inhibition Assay Protocol
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The α-glucosidase inhibition test was carried out in 96-well microplate using a modified procedure of Bilal et al.35 . The reaction mixture contained 50 μL of sodium phosphate buffer (100 mM, pH 6.8), 10 μL of α-glucosidase and 20 μL of plant (85.73 µg/ml)/fungal extract (69.72 µg/ml) as test sample; samples were thoroughly mixed in a 96-well microplate and were incubated at 37 °C for 30 minutes. However, in case of control, inhibitor was not added. The reaction was initiated by adding 20 μl of pNPG of varying concentration of 0.25–8 mg/mL, prepared in sodium phosphate buffer (100 mM, pH 6.8) as a substrate and incubated for another 15 minutes at 37 °C. The reaction was terminated by adding 50 μl of sodium carbonate. The absorbance was recorded at 405 nm by a 96-well micro plate reader (Bio-Rad, India). Acarbose (55.29 µg/ml) was used as a reference standard inhibitor.
The percentage of inhibition of both enzymes was calculated using the following equation: where Acontrol is the activity of enzyme without the inhibitor and Asample is the enzyme activity with the test sample solution as inhibitor at different concentrations. The amount of the inhibitor required for inhibiting 50% of the enzyme activity under the standard assay conditions was used to determine IC50 value.
The percentage of inhibition of both enzymes was calculated using the following equation: where Acontrol is the activity of enzyme without the inhibitor and Asample is the enzyme activity with the test sample solution as inhibitor at different concentrations. The amount of the inhibitor required for inhibiting 50% of the enzyme activity under the standard assay conditions was used to determine IC50 value.
8
Cell Proliferation and Clonogenicity Assays
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Cell proliferation was measured by WST-1 assay (DaeilLab, Seoul, Korea). In brief, cells were seeded at 3×103 viable cells/well onto 96-well microtiter plates in a final volume of 100 μL/well. Cells were incubated with WST-1 at 37°C for 2 hours and optical density (OD) values at 450 nm were recorded at days 0, 3, and 5 using a 96-well microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA). The experiment was performed in triplicate.
In order to examine clonogenicity, cells were seeded into 60-mm dishes (0.02-0.1×104 cells/well) and cultured for 3 weeks. Colonies formed in each well were fixed with H2O containing 10% methanol and 10% acetic acid, stained with 0.5% crystal violet, and then counted visually. A three-dimensional clonogenic growth assay was performed using a soft agar culture system. Briefly, cells were mixed in 0.3% agar containing 10% FBS and layered on top of the base layer of 0.6% agar in a 60-mm dish (3×103 cells/dish). After a four-week incubation in a humidified CO2 incubator, colonies were counted visually after staining with 0.5% crystal violet. Each cell group was cultured in triplicate, and colonies greater than 1.5 mm in diameter were scored visually.
In order to examine clonogenicity, cells were seeded into 60-mm dishes (0.02-0.1×104 cells/well) and cultured for 3 weeks. Colonies formed in each well were fixed with H2O containing 10% methanol and 10% acetic acid, stained with 0.5% crystal violet, and then counted visually. A three-dimensional clonogenic growth assay was performed using a soft agar culture system. Briefly, cells were mixed in 0.3% agar containing 10% FBS and layered on top of the base layer of 0.6% agar in a 60-mm dish (3×103 cells/dish). After a four-week incubation in a humidified CO2 incubator, colonies were counted visually after staining with 0.5% crystal violet. Each cell group was cultured in triplicate, and colonies greater than 1.5 mm in diameter were scored visually.
9
Silibinin Cytotoxicity Assay in RCC
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RCC cells were plated into 96-well culture plates and treated with various concentrations of silibinin for 24 h. The supernatant of each well was replaced with fresh medium containing 10% MTT (5 mg/ml) and incubated for a further 4 h. Subsequently, the medium was removed and 150 μl DMSO was added to each well. A 96-well microplate reader (Bio-Rad Laboratories, Inc.) was used to detect the absorbance at 570 nm.
10
Antiproliferative Effects of SB on PCa Cells
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Briefly, PCa cells were plated into 96-well plates at a density of 5.0×104 cells/ml and treated with different concentrations (0, 25, 50, 75, 100, 125, 150, 175, 200 µM) of SB for 24 or 48 h. DMSO was used as a negative control. Following the incubation, MTT (150 μl) was added/well for a further 4-h incubation at a temperature of 37°C. DMSO was subsequently added/well to dissolve the purple formazan crystals, and cell proliferation was measured using a 96-well microplate reader (Bio-Rad Laboratories, Inc.) at a wavelength of 490 nm. The equation of MTT assay was:
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