Human kidney HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The 293T cells were grown in DMEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and penicillin–streptomycin. Cells were incubated at 37 °C in a humidified atmosphere with 95% air/5% CO2. HEK293T cells (5 × 103) were incubated in 96-well plates to measure cell viability using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). After overnight incubation, the medium was changed to fresh medium containing CPT at doses of 50~0.1 μM in three wells each. Then, the cells were incubated for 24, 48, or 72 h and photographed under a phase-contrast microscope. We added 100 μL of fresh medium and 10 μL of CCK-8 solution to each well. After incubation for 3 h at 37 °C, the optical density (OD) at 450 nm was measured using a Spectramax i3x instrument (Molecular Devices, San Jose, CA, USA). The cell viability was calculated as follows: cell viability (%) = (OD (experiment) − OD (blank))/(OD (control) − OD (blank)) × 100%
Spectramax i3x instrument
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax i3x is a multi-mode microplate reader that provides flexible, high-performance detection capabilities. It features a monochromator-based optical system that enables absorbance, fluorescence, and luminescence measurements in a single instrument.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
Protein assay dye, by Bio-Rad (1 mentions)
3 mm tungsten carbide bead, by Qiagen (1 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Edta solution, by Thermo Fisher Scientific (1 mentions)
Mouse mpo elisa kit, by Hycult Biotech (1 mentions)
V plex plus proinflammatory panel 1 mouse kit, by Mesoscale (1 mentions)
Meso sector s 600 instrument, by Mesoscale (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Streptavidin hrp, by R&D Systems (1 mentions)
Heparin from porcine intestinal mucosa, by Merck Group (1 mentions)
High binding eia ria plates, by Corning (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
4 protocols using spectramax i3x instrument
1
Evaluating CPT Cytotoxicity in HEK293T Cells
2
Cell Proliferation Assay with CCK-8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HCC cells were seeded into 96-well plates at a density of 3 × 103 cells per well. Subsequently, 10 μL of CCK-8 solution (Dojindo, Tokyo, Japan) was added to each well at 0, 24, 48, and 72 h, followed by a 2-hour incubation period. The absorbance of the cells at 450 nm was then measured using a SpectraMax i3x instrument (Molecular Devices, USA). After 72 h, the proliferation curve of the cells was constructed based on the absorbance values.
3
Measuring Mucosal Inflammation Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total 6 mm pieces of distal colon or terminal ileum were placed in T-per buffer (Thermo Fisher Scientific) containing 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), 0.5 M EDTA solution (Thermo Fisher Scientific), and two 3 mm tungsten carbide beads (Qiagen). Tissues were disrupted in TissueLyser II (Qiagen) for 10 min at an oscillation frequency of 27.5 Hz. Generated tissue homogenates were centrifuged at 15000 rcf for 10 min at 4 °C and the supernatants were collected into deep 96-well plates. Protein assay dye (BioRad) was used to quantify total protein content using Bradford protein assay according to the manufacturer’s instructions. Cytokine concentrations were measured using V-PLEX Plus Proinflammatory Panel 1 mouse kit according to the manufacturer’s instructions (Meso Scale Diagnostics). Absorbance was measured on the Meso SECTOR S600 instrument (Meso Scale Diagnostics). Myeloperoxidase (MPO) activity was measured using a mouse MPO ELISA kit according to the manufacturer’s instructions (Hycult Biotech). Absorbance was measured on the SpectraMax i3x instrument (Molecular Devices). Data analysis was performed using GraphPad Prism™ (GraphPad Software, Inc.). Cytokine and MPO levels were normalized to total protein content.
4
Biotinylated GAG Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heparin from porcine intestinal mucosa (Merck), HS from bovine kidney (Merck), and chondroitin sulfate-A (CS) (Merck) were biotinylated as previously described in (46 (link)), using the EZ-Link Hydrazine-LC-Biotin kit (Thermofisher). Solid-phase binding assays were performed essentially as described previously (47 (link)). Recombinant proteins, either CXCL4, CXCL17 (24–119), SUMO3-CXCL17 (24–119), or SUMO3-CXCL17 truncation mutants, and SUMO3-tag were immobilized on 96-well EIA/RIA high binding plates (Corning) in coating buffer 20 mM Na2CO3, pH 9.6, overnight at RT. Wells were rinsed with 10 mM NaOAc, 150 mM NaCl, and 2% Tween-20, pH 6.0, and blocked with PBS and 5% BSA at 37˚C for 90 min. Biotinylated heparin, HS, or CS was added at 1 µg/mL or as otherwise indicated, and bound for 4 h at RT. Plates were washed with PBS, and the bound GAG was probed by 1:400 streptavidin-HRP (R&D Systems), and subsequent incubation with TMB substrate (ThermoFisher) for 10 min. Reaction was stopped with 0.2 M H2SO4, and OD450nm was measured with a SpectraMax i3x instrument (Molecular Devices). Background signals were corrected against blank wells, and data were analyzed and fit to non-linear hyperbola where X is concentration, to permit calculation of Bmax and KD values for GAG interaction with chemokine using Prism 9.2 (GraphPad, San Diego, CA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!