Mitochondrial activity quantification as a measure of cell viability was performed via MTT assay in 96-well cell culture microplates using Caco-2 (human colorectal adenocarcinoma) cells. Caco-2 cells were seeded at a density of 4 × 104 cells/well. Firstly, serial dilution was made of the following stock concentrations: 325 µg/mL ciprofloxacin solution, 1570 µg/mL SN-5 solution, 1570 µg/mL SN-10 solution, or 1520 µg/mL NF-5 solution, and then the cells were incubated with the mentioned materials at 37 °C for 24 h. Later, 20 μL of MTT reagent was added to each well. After an additional incubation at 37 °C for 4 h, 100 µL sodium dodecyl sulfate solution (10% in 0.01 M HCI) was added, and the cells were incubated overnight. The cytotoxicity of the compounds was then determined with an EZ READ 400 ELISA reader (Biochrom, Cambridge, United Kingdom) by measuring the OD at 550 nm (ref. 630 nm). The assay was repeated four times for each concentration.
Ez read 400 elisa reader
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The EZ READ 400 ELISA reader is a compact and user-friendly instrument designed for performing enzyme-linked immunosorbent assays (ELISAs). It measures the absorbance of samples in microplate wells, which is a key step in ELISA protocols.
Automatically generated - may contain errors
Lab products found in correlation
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (2 mentions)
Rpmi2650 cells, by American Type Culture Collection (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Lipopolysaccharide lps, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (1 mentions)
Spectrum one ftir spectrometer, by PerkinElmer (1 mentions)
Anaerobic jar, by Thermo Fisher Scientific (1 mentions)
7 protocols using ez read 400 elisa reader
1
Mitochondrial Viability Assay for Caco-2 Cells
2
Cytotoxicity Screening of Compounds
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Mitochondrial activity as a measure of cell viability was performed by MTT assay in 96-well cell culture microplates using RPMI 2650 cells (human nasal septum epithelial squamous carcinoma cells, obtained from American Type Culture Collection, ATCC, Manassas, VA, USA). RPMI 2650 cells were seeded at a density of 4 × 104 cells/well. First, serial dilution was made of the following stock concentrations: 4.11 mg/mL HPβCD_MEL-P_PVA_spd or 2.99 mg/mL αCD_MEL-P_PVA_spd or 5 µg/mL Lipopolysaccharide (LPS; ThermoFisher Scientific, Waltham, MA, USA), then the cells were incubated at 37 °C for 24 h. Later, 20 μL of thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After an additional incubation at 37 °C for 4 h, sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO, USA) solution (10% in 0.01 M HCI) was added and were incubated overnight. Cytotoxicity of the compounds was then determined by measuring the OD at 550 nm (ref. 630 nm) with EZ READ 400 ELISA reader (Biochrom, Cambridge, UK). The assay was repeated four times for each concentration.
3
Cytotoxicity Evaluation of Compounds 1-8
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For the determination of cytotoxic concentration 50% (CC50), a cell viability test was performed as an MTT assay using the Vero cell line. Cells were seeded at a density of 4 × 104 cells/well. After an overnight period, fresh medium was complemented with serial 2-fold dilutions of all compounds 1 –8 . The cells were incubated at 37 °C for 24 h. Later, 20 μL of thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After an additional incubation at 37 °C for 4 h, sodium dodecyl sulphate (Sigma-Aldrich, St. Louis, MO, USA) solution (10% in 0.01 M HCI) was added and incubated overnight. CC50 was then determined by measuring the OD at 550 nm (ref. 630 nm) with the EZ READ 400 ELISA reader (Biochrom, Cambridge, UK). The assay was replicated four times for each concentration.
4
ELISA Detection of Cryptosporidium Antigen
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A commercial ELISA kit, Cryptosporidium II (Techlab) was used to detect Cryptosporidium antigens from samples corresponding to trial days -1 (baseline), 12, 4, 6, 19–24 days (follow up 1) and 41–55 (follow up 2) following manufacturer’s instructions. The test was carried out in batches on fecal samples previously frozen at -80°C. Samples and reagents were brought to room temperature prior to testing. Results were read at a dual wavelength of 450–620 nm using a Biochrom EZ Read 400 ELISA reader (Biochrom, Cambridge, UK) with samples having optical density (OD) of ≥0.090 considered positive and OD<0.090 was considered negative. Positive and negative controls were run within each batch of testing. ELISA data from different plates then normalized.
5
Cytotoxicity Evaluation of Nanoparticles
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MTT assay was performed for the evaluation of the cytotoxic effect of NPs on the cells. A549 cells (adenocarcinoma human alveolar basal epithelial cells) were used as a model for alveolar type II cells, as these cells predominantly constitute the pulmonary alveolar epithelia [25 (link)]. For this purpose, A549 cells were seeded and cultured at a density of 4 × 104 cells/well in 96-well culture microplates. Later, the formulations, namely INH, blank CS/HA NPs, blank MC/HA NPs, INH-CS/HA NPs, and INH-MC/HA NPs, were added in different concentrations to the cultures and incubated for 24 h at 37 °C. After incubation for 24 h, 20 μL of MTT was added to each well, and the culture plates were again incubated for 4 h. Next, 100 μL of sodium dodecyl sulfate solution (10% in 0.01 M HCl) was added to the well plates to dissolve the formed formazan crystals. OD was measured using an EZ READ 400 ELISA reader (Biochrom, Cambridge, UK) at 550 nm (ref. 630 nm). Untreated cells with 100% viability were used as a control. All the evaluations were performed in triplicate. The relative cell viability was calculated using the following Equation (3),
6
Analytical Techniques for Compound Characterization
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Nuclear magnetic resonance (NMR) spectra were recorded on a 500 MHz Fourier transform (FT)-NMR spectrometer (Varian ECA 500 JOEL, Japan). Infrared (IR) spectra were obtained using a PerkinElmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). Mass spectra were obtained using an electrospray ionization mass spectrometry (ESI-MS) (ultra-performance liquid chromatography MS/MS TQD type, Waters). 2,2-diphenyl-1-picrylhydrazyl and SOD assays were measured on a Biochrom Ez Read 400 ELISA Reader.
7
Multi-Instrumental Characterization of Chemical Structure
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For the characterization of chemical structure, this study uses a 500 MHz FT-NMR spectrometer (Varian ECA 500 JOEL, Japan) to record the NMR spectra, a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England) to determine IR spectra, ES-MS Spectrometer (UPLC MS/MS TQD type, Waters) to get Mass Spectra data, and a UV spectrophotometer (Shimizu, Japan) to provide UV spectra. Meanwhile, the instrument for the antibacterial testing was measured on Biochrom EZ Read 400 Elisa Reader. Other instruments that support the test are incubator Memmert, autoclave machine HVE-50 Hirayama, laminar airflow, and anaerobic jar (for anaerobic condition, Oxoid).
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