Cell viability was performed by MTT assay. MTT powder was dissolved in phosphate-buffered saline (PBS) and prepared as 50 mg/mL stock. Sk-Hep1 or Hep3B cells (3 × 104) were placed in a 96-well plate overnight and incubated with and without fluoxetine at the final concentration (10, 20, 30 and 40 µM) for 24 h or 48 h. Media were then refreshed and incubated with 100 µL MTT medium combo solution per well (MTT final concentration was 5 mg/mL) for 4 h. Finally, MTT solution was replaced by 100 µL DMSO per well and the absorbance ratio was measured by Tecan Sunrise Absorbance Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland) at 570 nm [33 (link)].
Sunrise absorbance microplate reader
Manufactured by Tecan
Sourced in Switzerland, Austria, United States, United Kingdom, Germany
The Sunrise absorbance microplate reader is a versatile laboratory instrument designed to measure the absorbance of samples in microplates. It provides highly accurate and reproducible absorbance measurements across a wide range of wavelengths, enabling researchers to quantify the concentration of various biomolecules and perform diverse assays.
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Lab products found in correlation
Mtt reagent, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Mtt solution, by Merck Group (3 mentions)
Mtt assay, by Merck Group (2 mentions)
Sheep blood, by Merck Group (2 mentions)
Abts solution, by Roche (2 mentions)
96 well plate, by BD (2 mentions)
Cck 8 assay, by Dojindo Laboratories (2 mentions)
Hydrospeed microplate washer, by Tecan (2 mentions)
Abts solution, by Euromedex (2 mentions)
High binding 96 well elisa plates, by Corning (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Docetaxel, by Sanofi (2 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by American Type Culture Collection (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
Cobas probnp 2, by Roche (1 mentions)
Microcon centrifugal filter, by Merck Group (1 mentions)
L3771, by Merck Group (1 mentions)
99 protocols using sunrise absorbance microplate reader
1
Evaluating Fluoxetine's Impact on Cell Viability
2
Oxidation-Reduction Assay for Peroxides
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The PAB assay followed the standard protocol described elsewhere in detail (18 (link)). The assay measures differences in two ongoing reactions in the same sample: oxidation of chromogen 3,3’,5,5’-tetramethylbenzidine (TMB) by peroxides and reduction of coloured cation by antioxidants. The absorbance was measured colourimetrically on a Sunrise absorbance microplate reader at 450 nm (cut-off at 570 nm) (Tecan Group Ltd, Männedorf, Switzerland). The results are presented as arbitrary Hamidi-Koliakos (HK) units (percentage of hydrogen peroxide in the standard solution).
3
Fibrinogen γ' Sandwich ELISA Protocol
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The fibrinogen γ’ assay was constructed as a noncompetitive sandwich ELISA using fibrinogen γ’ mAb as capture antibody and biotinylated polyclonal rabbit antihuman fibrinogen as detection antibody.
Maxisorp ELISA plates (Thermo Fisher Scientific) were coated with 2 μg/mL fibrinogen γ’ mAb in coating buffer overnight at 4 °C in a humidity chamber. Plates were washed 4 times in washing buffer and blocked for 20 minutes in blocking buffer. Samples, calibrators, and controls were diluted in dilution buffer and incubated for 1 hour at RT with stirring, followed by washing 4 times in washing buffer. Biotinylated polyclonal rabbit antihuman fibrinogen was added in 1:500 dilution and incubated for 1 hour at RT with stirring. Following washing for 4 times, HRP-conjugated streptavidin (Invitrogen) diluted to 1:3000 was added and incubated with stirring for 30 minutes at RT. Plates were washed 4 times and visualized by incubating with 3,3’,5,5’-tetramethylbenzidine staining substrate (ECO-TEK Kementec) for 15 minutes protected by light. Next, color development was terminated by adding 0.2 M H2SO4. Finally, the plates were read at 450 nm, with 650 nm as a reference, using a Sunrise absorbance microplate reader (Tecan Group Ltd).
Maxisorp ELISA plates (Thermo Fisher Scientific) were coated with 2 μg/mL fibrinogen γ’ mAb in coating buffer overnight at 4 °C in a humidity chamber. Plates were washed 4 times in washing buffer and blocked for 20 minutes in blocking buffer. Samples, calibrators, and controls were diluted in dilution buffer and incubated for 1 hour at RT with stirring, followed by washing 4 times in washing buffer. Biotinylated polyclonal rabbit antihuman fibrinogen was added in 1:500 dilution and incubated for 1 hour at RT with stirring. Following washing for 4 times, HRP-conjugated streptavidin (Invitrogen) diluted to 1:3000 was added and incubated with stirring for 30 minutes at RT. Plates were washed 4 times and visualized by incubating with 3,3’,5,5’-tetramethylbenzidine staining substrate (ECO-TEK Kementec) for 15 minutes protected by light. Next, color development was terminated by adding 0.2 M H2SO4. Finally, the plates were read at 450 nm, with 650 nm as a reference, using a Sunrise absorbance microplate reader (Tecan Group Ltd).
4
Viability Assessment of Dental Pulp Stem Cells
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Disc specimens (6 mm in diameter and 0.6 mm in thickness) were prepared (n = 5) and placed into a 96-well plate containing 200 µL of fresh DPSCBM. A standard tissue culture plastic was used as a control. The hDPSCs at Passage 4 were seeded at a density of 5 × 103 cells/well. The cells were cultured for 3 days at a controlled temperature of 37 °C and a 5% CO2 humidified atmosphere. After 3 days of culture, an MTT viability assay was performed. DPSCs were incubated with a 0.2% 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 4 h. The reaction was stopped with 200 µL of dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and 25 µL of a glycine buffer (Research Organics, Cleveland, OH, USA). The end product’s color at an absorbance of 620 nm was measured using a spectrophotometer (Sunrise Absorbance Microplate Reader, Tecan Group Ltd., Männedorf, Switzerland). The results were reported as the relative cell viability (%) compared with the control using the following equation [33 (link)]:
where is the optical density.
where is the optical density.
5
Evaluating NPC Viability with MTT Assay
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the activity of NPCs. NPCs cells were seeded into 96-well plates (1 × 104 cells/well), incubated overnight at 37 °C, 5% CO2 incubator for 24 h. Then, NPCs were exposed to IL-1β, cyanidin and CQ, as described above. After indicated treatments, cells were washed with fresh medium and added to MTT solution (Sigma-Aldrich, St Louis, MO, USA) and incubated for 4 h at room temperature. After aspirating the MTT solution, the cells were incubated with 100 μL DMSO for 5 min at room temperature. The optical density values of the plate were measured at 490 nm on a Tecan Sunrise Absorbance Microplate Reader (Tecan Group, Switzerland).
6
SRB Assay for Cell Proliferation
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A sulforhodamine B colorimetric (SRB) assay was performed to assess cell proliferation based on the cellular protein content. The siRNA transfected cells were re-seeded in a 96-well plate (2×102 cells/well) and allowed to grow for 24–96 h. Cell monolayers were fixed with 10% trichloroacetic acid at 4°C for 1 h and then stained with 0.4% SRB in 1% acetic acid for 30 min in the dark. Next, the unbound dye was removed by washing for 5 times (1 min/each) with 1% acetic acid. The protein-bound dye was dissolved in 200 µl of 10 mM Tris Base solution for 1 h. The absorbance was measured at a wavelength of 540 nm using a Sunrise™ absorbance microplate reader (Magellan™ data analysis software version 6.6.0.1; Tecan Group Ltd., Männedorf, Switzerland). The experiment was performed in triplicate and repeated three times.
7
Evaluating IL-1β and ANI Effects on Cell Viability
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HPNCs cells were seeded into 96-well plates (1 × 106 cells/well), incubated overnight at 37°C, 5%CO2 incubator. Then, HPNCs were exposed to IL-1β (0–100 ng/ml) or ANI (0–200 μM) for 48 h. After treatments, cells were washed with fresh medium and added to MTT solution (Sigma-Aldrich, United States) and incubated for 4 h at room temperature. After aspirating the MTT solution, the cells were incubated with 100 μl DMSO for 5 min at room temperature. The optical density values of the plate were measured at 490 nm on a Tecan Sunrise Absorbance Microplate Reader (Tecan Group, Switzerland).
8
Cellular Viability and Proliferation Assay
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Viability and proliferation of cells (15 × 103 cells/well) were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay 48 h and 72 h after transfection with miRNA mimics. Of the MTT solution 20 µL (final concentration 0.5 mg/mL; ATCC®) was added to each well containing GIST-T1 cells and the plate was incubated at 37 °C for 2 h. Formed formazan crystals were dissolved in 200 µL of DMSO (Carl Roth®, Karlsruhe, Germany) at 37 °C for 15 min. Optical density values at 570 nm (with a reference filter of 620 nm) were measured using a Sunrise absorbance microplate reader (Tecan Trading AG, Mannedorf, Switzerland).
9
MTT Assay for Cell Viability
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The cell suspension (1 × 105 cells/mL) was cultured in 24-well plates for 24 h. Cells were pretreated with or without α-boswellic acid (1–50 μM) for 1 h, then MTT solution was added and incubated for 4 h. Finally, 200 μL of DMSO was added to each well, and 180 μL was pipetted into 96 wells for absorbance measurement. Absorbance was measured at 550 nm using Tecan’s Sunrise absorbance microplate reader (Tecan Trading AG, Männedorf, Switzerland).
10
Phage Infection Dynamics in Bacteria
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For the host culture collapse studies, an overnight culture of bacteria was refreshed in LB medium and allowed to grow until reaching OD600 of 0.2. Then each, culture was infected with the phage at MOIs ranging from 0.0001 to 1. The OD600 measurement was made every 15 min till the lysis of bacterial culture was observed. Measurements were carried out with the use of Sunrise absorbance microplate reader and Magellan v 6.6 2009 software (Tecan Trading AG, Männedorf, Switzerland).
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