NCI‐H2452 cells stably transfected with UBQLN4 shRNAs were plated in 96‐well plates (5 * 103 cells·well−1 in triplicate) and treated with 4 μm , 2 μm , 1 μm , 0.5 μm , 0.25 μm , 0.125 μm , 0.0625 μm , 0.03125 μm , 0.015625 μm , or 0.007813 μm of camptothecin for 24 h. After 72 h, the number of viable cells was measured using the CCK8 method (Bimake, B34304). Briefly, CCK8 solutions were added, and the plates were incubated at 37 ℃ for 1 to 4 h, and then, the absorbance NCI‐H2452 cells were determined using a spectrophotometric plate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Spectrophotometric plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Spectrophotometric plate reader is a laboratory instrument designed to measure the absorbance or optical density of samples in a multi-well microplate format. It is capable of quantifying the amount of light absorbed by a sample, which can be used to determine the concentration or presence of various molecules or compounds.
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Lab products found in correlation
Cck 8, by Dojindo Laboratories (2 mentions)
B34304, by Selleck Chemicals (1 mentions)
Type a chondroitin sulfate, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Sodium formate, by Merck Group (1 mentions)
Tioso4, by Merck Group (1 mentions)
Potassium iodide ki, by Merck Group (1 mentions)
Amplex red sphingomyelinase assay kit, by Thermo Fisher Scientific (1 mentions)
D8418, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
14 protocols using spectrophotometric plate reader
1
Evaluating Camptothecin Cytotoxicity in NCI-H2452
2
DMMB Assay for Glycosaminoglycan Quantification
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Several protocols were combined and modified to generate a DMMB assay for our study [60 (link),61 (link),62 (link),63 (link)]. A mixture was prepared by dissolving 21 mg of DMMB (Sigma, 341088) in 5 mL of absolute ethanol, followed by the addition of 2 g of sodium formate (Sigma). Then, the solution was mixed with 800 mL of double distilled water. Formic acid (95%, Sigma) was added dropwise to the solution until pH was 1.5, and then, distilled water was added to make 1L of final solution.
The process involved transferring 50 µL of centrifuged and digested samples into a 96-well plate. To this, 250 µL of DMMB was added to each well, and the contents were mixed by pipetting up and down. After incubation at room temperature for 1 h, a spectrophotometric plate reader (Thermo Fisher) was used to measure optical density (OD) of each well at 525 nm. A linear standard curve was also generated using various known concentrations of type A chondroitin sulfate (from bovine trachea, Sigma-Aldrich), and the GAG content of the hydrogel samples was extrapolated from the standard curve.
The process involved transferring 50 µL of centrifuged and digested samples into a 96-well plate. To this, 250 µL of DMMB was added to each well, and the contents were mixed by pipetting up and down. After incubation at room temperature for 1 h, a spectrophotometric plate reader (Thermo Fisher) was used to measure optical density (OD) of each well at 525 nm. A linear standard curve was also generated using various known concentrations of type A chondroitin sulfate (from bovine trachea, Sigma-Aldrich), and the GAG content of the hydrogel samples was extrapolated from the standard curve.
3
Quantifying Oxidative Species in PAW
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H2O2 concentrations in PAW were quantified using the titanium oxysulfate (TiOSO4, Sigma-Aldrich, Arklow, Ireland) colorimetric method. A total of 100 µL of each sample of PAW was incubated with 10 µL TiOSO4 in the dark for ten minutes. Absorbance was read on a spectrophotometric plate reader (ThermoScientific, Waltham, MA, USA) at 405 nm wavelength. A standard curve of known H2O2 concentrations was included on each plate and used to convert absorbance into H2O2 concentration [18 (link)].
Total oxidative species in PAW were measured using the potassium iodide (KI, Sigma-Aldrich, Arklow, Ireland) colorimetric method. A total of 50 µL of PAW or H2O2 standard samples were mixed with 50 µL deionized water and 100 µL 1 M potassium iodide (Sigma-Aldrich, Arklow, Ireland), incubated for twenty minutes, and the absorbance was read at 390 nm wavelength [19 (link)].
Total oxidative species in PAW were measured using the potassium iodide (KI, Sigma-Aldrich, Arklow, Ireland) colorimetric method. A total of 50 µL of PAW or H2O2 standard samples were mixed with 50 µL deionized water and 100 µL 1 M potassium iodide (Sigma-Aldrich, Arklow, Ireland), incubated for twenty minutes, and the absorbance was read at 390 nm wavelength [19 (link)].
4
Cell Proliferation Assay Protocol
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For proliferation assay, the transfected GC cells were seeded in 96-well plates (2,000 cells per well) and maintained at 37°C and 5% CO2 for 4 days. The reaction was stopped by addition of 150 µL dimethyl sulfoxide, and cell proliferation was determined by measuring the optical density at 490 nm using a spectrophotometric plate reader (Thermo Fisher Scientific).
5
Quantifying ESC Proliferation Using CCK-8
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The proliferation of ESCs was determined using Cell Counting Kit (CCK)-8 assays. After being transfected with siRNAs, the ESCs were seeded in 96-well plates at a density of 1 × 104 cells in 100 μL of culture medium per well. After a culture time of 0, 24, 36, and 48 h, 20 μL of CCK-8 (Dojindo, Kyushu, Japan) was added for an additional 4 h of incubation. A spectrophotometric plate reader (Thermo Fisher Scientific, Inc., MA, USA) was used to read the absorbance at 450 nm. This experiment was performed with triplicate wells and independently repeated at least three times.
6
Quantitative Sphingomyelin Assay in Cells
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Mouse brain tissues (100 mg) or hCMEC/D3 cells were homogenized in 1 mL of 1 M NaOH, and the total lipids were extracted twice from the tissues or cells with 1 mL of chloroform/methanol (2:1) solution. The organic layer was dried under nitrogen gas and re-solubilized in 0.2 mL of chloroform containing 2% Triton X-100. The lipid extract was dried and re-suspended in 0.2 mL of H2O containing 2% Triton X-100. Prior to the assay of SM level, the enzyme solution (containing SMase 25 U, alkaline phosphatase 500 U, choline oxidase 25 U, peroxidase 1000 U, DAOS 0.73 mM, and 4-aminoantipyrine 0.73 mM) was prepared in 50 mL of reaction buffer (0.05 M Tris-HCl with 0.66 mM calcium chloride, pH 8.0). Next, 10 µL of the lipid extract was added to 100 µL of enzyme solution. After reacting at 37°C for 45 minutes, the absorption at 595 nm was measured on a spectrophotometric plate reader (Thermo Fisher Scientific; Zhang et al., 2011).
7
Quantifying Neuronal Sphingomyelinase Activity
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The activity of nSMase1 in the mouse brains and hCMEC/D3 cells was determined using the Amplex® Red Sphingomyelinase Assay Kit (Thermo Fisher Scientific, Cat# A12220) as described in the manual. The fluorescence of samples at 550 nm excitation and 590 nm emission was monitored at different times using a spectrophotometric plate reader (Thermo Fisher Scientific). nSMase1 activity was calculated from the slope of an intrinsic fluorescence vs. time graph and standardized per mg of protein (mU/h/mg protein).
8
hCMEC/D3 Cell Viability Assay
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Cell viability of hCMEC/D3 cells was detected by the methylthiazolyl tetrazolium (MTT) assay (Neves et al., 2021). Cells were plated in 96-well plates and pretreated with reagents described in the section “Cell culture and treatment.” The Endothelial Cell Medium was removed and cells were incubated with 0.5 mg/mL MTT (MilliporeSigma, Cat# M2003) for 4 hours at 37°C. The reaction was stopped by adding dimethyl sulfoxide (MilliporeSigma, Cat# D8418). The amount of MTT formazan product was determined by measuring the absorbance at 570 nm with a spectrophotometric plate reader (Thermo Fisher Scientific).
9
Hesperidin Cytotoxicity Evaluation
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HN6 and HN15 (5000 cells/well) cells were seeded in a 96-well culture plate overnight and then exposed to various doses (0, 6.25, 12.5, 25, 50, 100, and 200 µM) of hesperidin for 24, 48, or 72 h. Cell viability was determined by MTT assay as has been previously described [23 (link)]. The optical density (OD) of dissolved formazan dye was measured using a spectrophotometric plate reader (Thermo Fisher Scientific, Inc.) at 540 nm with a reference wavelength of 630 nm. The percent of cell viability (% of cell viability) was calculated as the sample/OD of control × 100.
10
MTT Assay for Cell Proliferation
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The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to examine the proliferation of plasmid transfected LoVo cells and untransfected LoVo cells. All cells were uniformly seeded into 96-well culture plates with a logarithmic growth phase at a concentration of 5 cells/µL in a volume of 100 µL per well. 48 h later, 20 µl of MTT (Kaiji, Nanjing, China) reagent (5 mg/mL) was added into each well, and the plates were incubated for 4 h at 37°C in 5% CO2. After discarding the mixture, 200 µL dimethylsulfoxide (Sigma, St. Louis, USA) was added and shaken for 10 min to dissolve the MTT. Then the optical density (OD) value was measured by a spectrophotometric plate reader (Thermo, Waltham, MA, USA) at 492 nm. Each group was comprised of eight duplicated wells, and the assay was performed three times independently.
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