Cell viability was determined via sulforhodamine B (SRB) assay. HepG2 cells were seeded in 96-well plates at a density of 1 × 104 cells and incubated for 24 or 48 h. After treatment with RA-XII, 100 µL of 10% TCA was added to each well and fixed in for 1 h. After washing three times with water, 100 µL of 4% SRB was added to each well and stained 15 min. Then, washed three times with 1% TCA, stained cells were dissolved with 10 mM unbuffered Trisbase (pH = 10.5) and were measured the absorbance at 540 nm using a microtitre plate reader (Bio-Rad, Hercules, CA, USA).
Microtitre plate reader
Manufactured by Bio-Rad
Sourced in United States
The Microtitre plate reader is a laboratory instrument designed to measure the absorbance or fluorescence of samples in a multi-well microplate format. It is commonly used for various assays, such as enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other applications that require the quantification of optical properties of samples in a high-throughput manner.
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Lab products found in correlation
Cell proliferation kit 2 xtt, by Roche (1 mentions)
Flat bottomed plates, by Corning (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Colorimetric assay kit, by Abcam (1 mentions)
Xcelligence dp device, by Roche (1 mentions)
Rtca software, by Roche (1 mentions)
E plate, by Roche (1 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (1 mentions)
14 protocols using microtitre plate reader
1
Cell Viability Determination via SRB Assay
2
Microbial Antibiotic Susceptibility Assay
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Minimal inhibitory concentration (MIC) assay was performed using microtitre plate method (Stubbings et al., 2004 (link)). Overnight cultures of MG1655 and Δ10 strains were diluted to 0.05 OD600. Appropriate dilutions of ciprofloxacin (ranging from 1 to 10 ng/ml, with increments of 1 ng/ml) were made in LB broth. 200 μl of the ciprofloxacin supplemented media a loaded on to 96 well microtitre plates. Two microlitres of the diluted cultures were inoculated into each of the well. The plates were grown at 37°C in a shaker incubator for 16 h. OD595 of each well was measured with a Biorad Microtitre plate reader. These experiments were done thrice in triplicates.
3
Quantifying Microbial Metabolic Activity
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The (XTT)-reduction assay has been used as a routine tool for the quantitative measurement of bacterial and fungal metabolic activity, growth and response to antimicrobial treatments78 (link)–82 (link). After peptide treatment, the medium was aspirated from each well to remove floating cells, and the wells were thoroughly washed twice with PBS. The assay was conducted as described by Barra et al.80 (link) with some modifications. Two hundred microlitres of XTT solution was added to each well, and the plate was incubated in the dark for 30 min at 37 °C. Changes in the absorbance of XTT were measured spectrophotometrically at 490 nm using a microtitre plate reader (Biorad). An XTT cell proliferation Kit II was purchased from Roche Diagnostics. Viability ratios were computed for each well with respect to their relative controls.
4
DTMUV Antibody ELISA in Ducks
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Serum samples from ducks were determined by an indirect ELISA test using the recombinant E protein of DTMUV, produced in E. coli BL21 (DE3), as antigen. The E protein was expressed in E. coli BL21 (DE3) using the PET 32a expression system (Novagen) and the product were purified by dialysis method. Ninety-six wells flat-bottomed plates (Corning Costar) were coated with recombinant E protein in 0.1 M carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. After blocking with 5% BSA in PBS, plates were incubated with duplicate twofold serial dilutions of test sera for 1 h at 37 °C. Rabbit anti-ducks IgG HRP (KPL) at a 1:2000 dilution was then added for 1 h at 37 °C, followed by the addition of the substrate 2 mm Sulfuric acid. Absorbance was determined at 450 nm using a Bio-Rad microtitre plate reader.
5
Cytotoxicity Evaluation of AANG on R-HepG2 Cells
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The MTT assay was used to determine the cytotoxicity of AANG on R‐HepG2 cells in vitro. In brief, R‐HepG2 cells (1 × 104 cells/well) were seeded on a 96‐well plate and serial dilutions of AA, NG or their combination with indicated concentration were added on the next day. After 24‐h of treatment, 30 microlitres of methyl‐thiazoldiphenyl tetrazolium (MTT) (5 mg/ml) was added to each well and incubated for 2 h at 37°C. The MTT solution was then replaced by 100 µl of dimethyl sulphoxide in each well and measured with a microtitre‐plate reader (Bio‐Rad) at 540 nm, and all data were calculated as percentage against the control.
6
NF-κB Transcription Factor Binding Assay
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MCF-7 and MDA-MB-231 cells were treated with 6 (0, 1 nM, 2.5 nM, 5 nM) for 4 h. Subsequently, nuclear proteins were isolated using a nuclear extract kit (Active Motif). Extracts were then assayed for NF-κB subunit DNA binding with a TransAM NF-κB family kit according to the manufacturer’s protocol. The consensus oligonucleotide used for NF-κB binding was 5′-GGGACTTTCC-3′, and specific NF-κB proteins bound to the oligonucleotide were identified using primary antibodies for NF-κB subunits p50, p52, p65, c-Rel and RelB, followed by detection with an HRP-conjugated secondary antibody. The absorbance at 450 nm (A450) was read on a microtitre plate reader (Bio-Rad), and readings were compared with the untreated controls.
7
Antiproliferative Activity of SIS3 on B16F10 Cells
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The MTT assay was used to determine the antiproliferative activity of SIS3 on B16F10 cells in vitro. In brief, cancer cells (1 × 104 per well) were seeded on a 96-well plate and serial concentrations of SIS3 (0, 2.5, 5, 10, 15, 20 μM) were added on the next day. After 24-h of SIS3 treatment, 30 μl of MTT (5 mg ml−1) was added to each well and incubated for 2 h at 37 °C. The MTT solution was then replaced by 100 μl of dimethyl sulfoxide in each well and measured with a microtitre-plate reader (Bio-Rad) at 540 nm, and all data were calculated as a percentage against the control.
8
NDRV Sigma C Protein ELISA for Duck Serum
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Serum samples from ducklings were determined by an indirect ELISA test using the recombinant sigma C protein of NDRV, produced in E. coli BL21 (DE3), as antigen. The sigma C protein was expressed in E. coli BL21 (DE3) using the PET 32a expression system (Novagen, USA) and the recombinant product was purified by dialysis method. Ninety-six wells flat-bottomed plates (Corning Costar, USA) were coated with recombinant sigma C protein in 0.1 M carbonate/bicarbonate buffer (pH9.6) and incubated overnight at 4 °C. After blocking with 5% BSA in PBS, plates were incubated with duplicate twofold serial dilutions of test sera for 1 h at 37 °C. Rabbit anti-duck IgG HRP (KPL, USA) at a 1:2000 dilution was then added for 1 h at 37 °C, followed by the addition of the substrate 2 mM Sulfuric acid. Absorbance was determined at 450 nm using a Bio-Rad microtitre plate reader.
9
Sulforhodamine B Assay for Cell Viability
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Cell viability was determined via sulforhodamine B (SRB) assay. HepG2 cells were seeded in 96-well plates at a density of 1 × 104 cells and incubated for 24 or 48 h. After treatment with RA-XII, 100 μL of 10% TCA was added to each well and fixed in for 1 h. After washing three times with water, 100 μL of 4% SRB was added to each well and stained 15 min. Then washed three times with 1% TCA, stained cells were dissolved with 10 mM unbuffered Trisbase (pH = 10.5) and were measured the absorbance at 540 nm using a microtitre plate reader (Bio-Rad, Hercules, CA, USA).
10
CXCL1 Protein Binding ELISA
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Recombinant human CXCL1 was diluted to a final concentration of 10 μg/mL in 0.01 M phosphate- buffered saline pH 7.2 (PBS). One hundred microliters of the solution were used for coating each well of the ELISA plates (Maxisorp; Nunc, Roskilde, Denmark) at 4°C overnight. The plates were thereafter washed three times with 350 μl of washing solution (PBS). The plates were blocked at room temperature for 1 h with 200 μl of 3% (w/v) albumin from bovine serum (BSA) in PBS and then washed 3 times. Then 6.25 μg of HL2401 (control) or 6.25 μg of serum proteins were added to wells in triplicate. The plate was incubated at room temperature for 1 h. After removal of unbound material (4 washes with 0.05% Tween 20-PBS), 50 μl of IgG-HRP as the secondary antibody; diluted in PBS-T 1:1000 dilution Mouse IgG-HRP (Santa Cruz Biotech) was added to each well and incubated for 30 min at room temperature. After 4 repeated PBS-T washes, 100 μl of TMB Abcam high sensitivity was added to each well for 15 min at room temperature. The enzyme-substrate reaction was stopped by adding 100 μl of 2N sulfuric acid to each well. The optical density (OD450) was measured at 450 nm using a microtitre plate reader (Bio-Rad, Hercules, California, USA).
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