Microplate spectrophotometer

Manufactured by Bio-Rad

Sourced in United States, Japan

The Microplate spectrophotometer is a laboratory instrument designed to measure the absorbance of samples in a microplate format. It is used to quantify various biomolecules, such as proteins, nucleic acids, and small molecules, by detecting their optical properties.

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Lab products found in correlation

Methyl thiazolyl tetrazolium (mtt), by Merck Group (13 mentions) Super aquablue elisa substrate, by Thermo Fisher Scientific (9 mentions) High protein binding microtiter plates, by Corning (7 mentions) Cck 8 kit, by Dojindo Laboratories (6 mentions) Horseradish peroxidase hrp conjugated goat anti human igg antibody, by Jackson ImmunoResearch (4 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (4 mentions) Neopterin competitive enzyme immunoassay, by ALPCO (4 mentions) Dimethyl sulfoxide (dmso), by Merck Group (4 mentions) Human galectin 9 quantikine elisa kit, by R&D Systems (3 mentions) Tmb substrate reagent set, by BD (3 mentions) Fetal bovine serum (fbs), by Welgene (3 mentions) Milliplex analyst software, by Merck Group (3 mentions) Elisa kit, by Jiangsu Meimian (3 mentions) Co2 incubator, by Thermo Fisher Scientific (3 mentions) Mtt reagent, by Merck Group (2 mentions) Triton x 100, by Merck Group (2 mentions) Fetuin, by Merck Group (2 mentions) Sigmafast opd, by Merck Group (2 mentions) Ap504p, by Merck Group (2 mentions) Luciferase reporter gene assay kit, by Beyotime (2 mentions)

182 protocols using microplate spectrophotometer

Cytotoxicity Evaluation of TiO₂NPs in MCF-7 Cells

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MCF-7 cells were seeded in 96 well microplates at a concentration of 5 × 10³ cells/well and stabilized in an incubator for 24 h. Successively, TiO₂NPs at two different concentrations (25 and 50 µg/mL) were added to cell media. After incubation times of 24 and 48 h, standard WST-8 assay (96992, Sigma Aldrich, Darmstadt, Germania) and the lactate dehydrogenase (LDH) leakage assay, using the CytoTox-ONE Homogeneous Membrane Integrity Assay reagent (G7890, Promega, Madison, WI, USA), were performed to conduct viability testing and to evaluate the membrane damage, respectively.
The WST-8 assay procedure that was used is described in our previous work [43 (link)].
The amount of lactate dehydrogenase (LDH), a soluble cytosolic enzyme released after cell lysis, was measured by reading absorbance at 490 nm using a Bio-Rad microplate spectrophotometer. The increase of the LDH activity in culture supernatant is proportional to the number of cells lysed. Data were expressed as mean ±SD. Mean values differences between cells treated and respective controls were considered statistically significant performing a t-student test (p-value < 0.05)

Cascione M., De Matteis V., Mandriota G., Leporatti S, & Rinaldi R. (2019). Acute Cytotoxic Effects on Morphology and Mechanical Behavior in MCF-7 Induced by TiO2NPs Exposure. International Journal of Molecular Sciences, 20(14), 3594.

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SARS-CoV-2 Antibody Binding Assay

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High-protein binding microtiter plates (Costar) were coated with recombinant SARS-CoV-2 proteins at 2 μg/ml in 1X PBS overnight at 4°C. Plates were washed the next morning with 1X PBS 0.05% Tween and blocked with 1X PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1:3 starting at 10 μg/ml and incubated for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody diluted 1:1000 (Jackson Immuno Research) was used to detect binding of mAbs, and plates were subsequently developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). To standardize the assays, control antibodies with known binding characteristics were included on each plate and the plates were developed when the absorbance of the control reached 3.0 OD₄₀₅ units. All experiments were performed in duplicate 2–3 times.

Dugan H.L., Stamper C.T., Li L., Changrob S., Asby N.W., Halfmann P.J., Zheng N.Y., Huang M., Shaw D.G., Cobb M.S., Erickson S.A., Guthmiller J.J., Stovicek O., Wang J., Winkler E.S., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Diamond M.S., Fremont D.H., Kawaoka Y, & Wilson P.C. (2021). Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets. Immunity, 54(6), 1290-1303.e7.

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Evaluating GIE's Effect on Cell Viability

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The effect of GIE on cell viability was evaluated by using a tetrazolium dye colorimetric assay as described by Tiamyom et al. [14 (link)] and Dunkhunthod et al. [24 (link)]. Briefly, RAW264.7 cells were seeded in a 96-well plate (2 × 10⁴ cells/well) and cultured for 24 h. Cells were treated with different concentrations of GIE for 24 h. Following treatment, the culture medium was removed, the MTT solution (0.5 mg/mL) (Cat. No. M6494, Invitrogen, Carlsbad, CA, USA) was added and further incubated at 37°C for 4 h. Subsequently, 150 μL DMSO was added to dissolve formazan crystal formed by viable cells, and absorbance was measured at 540 nm with a microplate spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Dunkhunthod B., Talabnin C., Murphy M., Thumanu K., Sittisart P, & Eumkeb G. (2021). Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages. Oxidative Medicine and Cellular Longevity, 2021, 8658314.

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Bacterial Growth Kinetics Measurement

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Fresh overnight bacterial culture was inoculated into a 50 mL centrifuge tube containing 10 mL NB or XVM2 media at an initial optical density of OD₆₀₀ = 0.05. At each time point, 200 µL bacterial culture of each strain was taken out and measured using microplate spectrophotometer (Bio‐Rad Laboratories, Hercules, CA, USA). The experiments were repeated at least twice in triplicate with similar results.

Zhang Y., Teper D., Xu J, & Wang N. (2019). Stringent response regulators (p)ppGpp and DksA positively regulate virulence and host adaptation of Xanthomonas citri. Molecular Plant Pathology, 20(11), 1550-1565.

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Quantifying Biofilm Formation in P. aeruginosa

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An overnight culture of P. aeruginosa was diluted 200‐fold with LB containing twofold serially diluted glucose or levofloxacin (OFLX) or horse serum (HoS), and 200 μl of the suspension was added into microplates. After incubation at 37°C for 24 hr without shaking, the planktonic cells were removed by saline washing, and the biofilm cells adhering to the wells were stained by crystal violet or XTT [2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide].
For crystal violet staining (She et al., 2018), 200 µl of 0.25% (wt/vol) crystal violet was added to each well and incubated at room temperature for 15 min. Unbound dye was removed by saline washing. The plates were allowed to air dry, and 95% ethanol was added to dissolve the bound dye. After incubation at room temperature for 20 min, the absorbance of the ethanol at 570 nm (A570) was measured by a microplate spectrophotometer (Bio‐Rad, USA).
For XTT staining assay (Psoter, Rosenfeld, De Roos, Mayer, & Wakefield, 2014), XTT was diluted with 1 × PBS (pH = 7.0) to a final concentration of 0.2 mg/ml and mixed with phenazine methosulfate (0.02 mg/ml). Then, 200 μl of this mixture was added to each well, and after incubation at 37°C for 3 hr in the dark, the absorbance at 490 nm (A490) was measured.

She P., Wang Y., Liu Y., Tan F., Chen L., Luo Z, & Wu Y. (2019). Effects of exogenous glucose on Pseudomonas aeruginosa biofilm formation and antibiotic resistance. MicrobiologyOpen, 8(12), e933.

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Mitochondrial Function Assays: CCO Activity and ATP Production

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To examine the effect of PBM on mitochondrial function, assays of CCO activity and ATP production were carried out as described previously [39 (link)]. Briefly, ATP concentration was determined using an ENLITEN® rLuciferase/Luciferin kit (FF2021, Promega) as described. Cytosolic protein samples (30 μg) were suspended in 100 μL of reconstituted rL/L reagent buffer. The reactions were measured in a microplate luminometer (PE Applied Biosystems) and values were calculated from the ATP standard curve. CCO activity was measured with a Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, Abcam) using an equal amount of protein sample from the mitochondrial fractions. The activities of immuno-captured CCO proteins within the 96-well microplate were determined colorimetrically by measuring the degree of cytochrome c oxidation at 550 nm absorbance on a microplate spectrophotometer (Bio-Rad). The values of ATP concentration and relative CCO activity were expressed as percentage changes versus sham GCI group for graphical depiction.

Wang R., Dong Y., Lu Y., Zhang W., Brann D.W, & Zhang Q. (2018). Photobiomodulation for Global Cerebral Ischemia: Targeting Mitochondrial Dynamics and Functions. Molecular neurobiology, 56(3), 1852-1869.

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Cell Viability Assay using CCK-8 Reagent

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PC cells were seeded into 96-well plates at a density of 2×10³ cells/well and incubated at 37°C for 6, 24, 48, 72 or 96 h. Following incubation, the medium was removed and 100 µl fresh DMEM containing 10 µl CCK-8 reagent (Wuhan Boster Biological Technology, Ltd.) was added to each well. The cells were subsequently incubated with 5% CO₂ at 37°C for 2 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate spectrophotometer (Bio-Rad Laboratories, Inc.).

Lei S., Zeng Z., He Z, & Cao W. (2021). miRNA-7515 suppresses pancreatic cancer cell proliferation, migration and invasion via downregulating IGF-1 expression. Oncology Reports, 46(3), 200.

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Quantitative ELISA for Galectin-9

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Stored plasma aliquots were measured for Gal-9 in duplicate using the solid-phase Human Galectin-9 Quantikine ELISA kit (R&D systems, MN, USA) according to manufacturer’s instructions. Optical density was read with a microplate spectrophotometer (Bio-Rad) and data analysis, including four parameter logistic standard interpolation, was carried out using MyAssays Ltd. data analysis. Average intra-assay coefficient of variation (CV) was 4.11% and inter-assay CV was 9.95%.

Premeaux T.A., Moser C.B., McKhann A., Hoenigl M., Laws E.I., Aquino D.L., Lederman M.M., Landay A.L., Gianella S, & Ndhlovu L.C. (2021). Plasma galectin-9 as a predictor of adverse non-AIDS events in persons with chronic HIV during suppressive antiretroviral therapy. AIDS (London, England), 35(15), 2489-2495.

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MTT Assay for Cell Viability

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The transfected HeP3B and sk-Hep-1 cells were cultured for 0, 1, 2, 3 or 4 days at 37°C, respectively. Twenty-microliter MTT (5 mg/mL) was then added into cells at different time points. After 4 h of incubation, 150 μL DMSO was added in cells. The absorbance value at 450 nm was measured by a microplate spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA)

Zhao W., Jiang X, & Yang S. (2020). lncRNA TUG1 Promotes Cell Proliferation, Migration, and Invasion in Hepatocellular Carcinoma via Regulating miR-29c-3p/COL1A1 Axis. Cancer Management and Research, 12, 6837-6847.

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HDF Proliferation Assay using CCK-8

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HDF proliferation was assessed using the cell counting kit-8 assay (CCK-8; Dojindo, Tokyo, Japan) according to the manufacturer's instructions. HDFs (5 × 10³ cells/well) were seeded onto 96-well plates. Following one day of culture, cell proliferation was measured for 6 days. Absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer (Bio-Rad, California, USA).

Qin F., Huang J., Zhang W., Zhang M., Li Z., Si L., Long X, & Wang X. (2020). The Paracrine Effect of Adipose-Derived Stem Cells Orchestrates Competition between Different Damaged Dermal Fibroblasts to Repair UVB-Induced Skin Aging. Stem Cells International, 2020, 8878370.

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