PGE2 inhibitory activity of LI13019F1 was evaluated in human PBMCs [28 (link)]. Briefly, an equal number of PBMCs were seeded in each well of a 96-well culture plate, and the cells were treated with different concentrations of LI13019F1 for 2 hr at 37°C in a CO2 incubator. Then, the cells were induced with 10 ng/mL LPS and incubated further for 4 hr. The cell culture wells received only 0.2% DMSO served as the vehicle control. The PGE2 was measured in the cell-free culture supernatant (clarified at 270 g, 5 min), using an ELISA kit. The PGE2 assay was performed following the manufacturer's instructions. Absorbance was measured at 412 nm in a microplate reader (Spectramax 2e; Molecular Devices, San Jose, CA).
Spectramax 2e
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax 2e is a microplate spectrophotometer designed for life science research applications. It measures the absorbance of samples in microplates, allowing for quantitative analysis of various biomolecules and cellular assays.
Automatically generated - may contain errors
Lab products found in correlation
Ab102500, by Abcam (1 mentions)
Human tnf α elisa kit, by R&D Systems (1 mentions)
Phusion hf buffer, by New England Biolabs (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Fastdna spin kit, by MP Biomedicals (1 mentions)
G box, by Syngene (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
8 protocols using spectramax 2e
1
Evaluation of PGE2 Inhibitory Activity
2
Quantifying Cartilage Matrix in Chondrocytes
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GAG staining with 1,9-dimethylmethlyene blue (DMMB) in chondrocytes was employed to evaluate whether LI13019F1 can modulate cartilage matrix production in IL-1β-induced HCH human primary chondrocytes [29 (link)]. Briefly, an equal number of HCH cells were seeded in each well of a 48-well cell culture plate. The cells were treated with 5 ng/mL of IL-1β in the presence or absence of an increasing concentration of LI13019F1 and incubated for 72 hr at 37°C in the presence of 5% CO2. Following the treatment period, the washed cells were lysed in cell lysis buffer, and an equal volume of cell lysates was reacted with the DMMB dye reagent (16 mg DMMB in 95 mL 0.1 M acetic acid containing 3.04 g glycine and 1.6 g NaCl). Immediately after the color development, the absorbance was read at 525 nm in a microplate reader (Spectramax 2e; Molecular Devices, San Jose, CA). Increasing concentrations of chondroitin sulfate A were run in parallel to construct a standard curve for the quantitative measurement of the ECM components present in the cell lysates.
3
Kinetic Characterization of Azole Biosynthesis
4
Measurement of TNF-α in PBMC Cultures
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An equal number of human PBMCs suspended in phenol red-free DMEM containing 1% FBS were plated in a 96-well flat-bottom culture plate. Cells were pretreated with different concentrations of LI13019F1 at 37°C for 2 hr in the presence of 5% CO2. After that, the cells were induced with 1 μg/ml LPS and incubated further for 4 hr. The vehicle control culture wells received only 0.1% (v/v) DMSO in DMEM-1% FBS. TNF-α present in the cell-free culture supernatants (clarified at 270 g, 10 min) was quantified using a Human TNF-α ELISA kit (R&D Systems, Minneapolis, MN). The assay was performed following the manufacturer's instructions. Absorbance was measured at 450 nm in a microplate reader (Spectramax 2e; Molecular Devices, San Jose, CA). An increasing concentration of TNF-α was run in parallel to construct a standard curve for the quantitative measurement of TNF-α present in the culture supernatants.
5
Metagenomic DNA Isolation and 16S rRNA Sequencing
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Isolation of the metagenomic DNA from sa-um samples were carried out using the Fast DNA spin kit (MP Biomedical, USA) and quantified by microplate reader (Spectra Max 2E, Molecular Devices, USA). The V4 hyper variable region of the 16S rRNA gene was amplified by 10 pmol/μl of each forward 515F (5′-3′) and reverse 806R (5′-3′) primers. The amplification mix contained 40 mM dNTPs (NEB, USA); 5× Phusion HF buffers (NEB, USA); 2 U/μl F-540 Special Phusion HS DNA Polymerase (NEB, USA); and 5 ng DNA and Milli-Q water to make up 30 μl total volume. PCR conditions consisted of initial denaturation at 98 °C for 30 s followed by 30 cycles of 98 °C for 10 s, 72 °C for 30 s, and a final extension at 72 °C for 5 s. Paired end Illumina MiSeq sequencing (2 × 250 bp) was performed at SciGenom Lab, Cochin, India (De Mandal et al. 2016 (link), 2017 (link)).
6
Chondrocyte Survival Modulation by LI13019F1
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LI13019F1 was evaluated for its ability to modulate the survival of IL-1β-induced HCH human primary chondrocytes. Briefly, an equal number of HCH cells were plated in each well of a 96-well cell culture plate and incubated overnight at 37°C in the presence of 5% CO2. The next day, the culture medium was replaced with fresh medium containing IL-1β (5 ng/mL) and allowed to grow for 72 hr in the presence or absence of increasing concentrations of LI13019F1. The vehicle control cultures received only 0.2% DMSO (v/v) containing growth medium. Following the treatment period, the culture wells were washed with serum-free and phenol red-free DMEM. Finally, an equal volume of XTT reagent working solution was added to each well and incubated in the dark at 37°C. The absorbance at 490 nm with a reference wavelength of 630 nm was determined using a microplate reader (Spectramax 2e; Molecular Devices, San Jose, CA). The absorbance values in the vehicle control wells were considered as 100 percent cell viability.
7
Efficient DNA Extraction from Soil Samples
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Total DNA was extracted from 2 to 5 g of collected sample using Fast DNA™ Spin kit for soil (MP Biomedicals, USA). The DNA concentration was quantified using a microplate reader (SpectraMax 2E, Molecular Devices, USA). Agarose gel electrophoresis of the community DNA was carried out to check the quality of the DNA, stained with ethidium bromide and visualized under gel documentation system (G-Box, SynGene, USA).
8
Quantification of LTB4 in PMNs
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An equal number of PMNs seeded in each well of a 96-well microplate was treated with different concentrations of LI13019F1. The vehicle control wells received 0.2% DMSO (v/v). The plate was incubated in a CO2 incubator at 37°C for 2 hr. The cells were induced with 10 µM A23187 for a further 10 min at 37°C in the presence of 5% CO2. An ELISA kit measured the LTB4 content in the cell-free culture supernatants (clarified at 270 g, 5 min), following the manufacturer's instructions (R&D Systems, Minneapolis, MN). The assay utilized the competitive antigen-antibody binding principle. Following the substrate reaction, the absorbance of the developed color was measured at 450 nm in a microplate reader (Spectramax 2e; Molecular Devices, San Jose, CA). The amount of LTB4 present in the supernatants was quantified, comparing the optical density values with a standard curve plotted against the known concentrations of the analyte.
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