Evaluation of Inflammatory Markers in IEC-6 Cells

IEC-6 cells were plated into 96-well plates (2 × 10³ cells/well) and treated with the peptides in inflammatory conditions, as previously described, for 24 h in order to evaluate COX-2, iNOS, HO-1, and SOD expression and nitrotyrosine formation. For this analysis, the cells were collected and washed with phosphate-buffered saline (PBS). Fixing solution was added to cells for 20 min and then incubated in fix/perm solution for a further 30 min. Anti-COX-2 (BD Transduction Laboratories, Milan, Italy), anti-iNOS (BD Transduction Laboratories, Milan, Italy), anti-HO-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-SOD (Santa Cruz Biotechnologies, Dallas, TX, USA), and anti-nitrotyrosine (Merck Millipore, Milan, Italy) antibodies were then added for 1 h. The secondary antibody, in fixing solution, was added to the cells and cell fluorescence was then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy) [35 (link)].

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Basilicata M.G., Pepe G., Rapa S.F., Merciai F., Ostacolo C., Manfra M., Di Sarno V., Autore G., De Vita D., Marzocco S, & Campiglia P. (2019). Anti-Inflammatory and Antioxidant Properties of Dehydrated Potato-Derived Bioactive Compounds in Intestinal Cells. International Journal of Molecular Sciences, 20(23), 6087.

Publication 2019

Anti-COX-2 anti-iNOS anti-HO-1 anti-SOD anti-nitrotyrosine FACSscan Cell Quest software (version 4;

Antibodies Antibody Cell Cox 2 Fluorescence Inflammatory Inos Nitrotyrosine Peptides Perm Phosphate Saline

Corresponding Organization : European Biomedical Research Institute of Salerno

Other organizations : University of Naples Federico II, University of Basilicata, Sapienza University of Rome

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Variable analysis

independent variables

Peptides

dependent variables

COX-2 expression
INOS expression
HO-1 expression
SOD expression
Nitrotyrosine formation

control variables

Inflammatory conditions
Cell type (IEC-6 cells)
Cell density (2 × 10^3 cells/well)
Incubation time (24 h)
Phosphate-buffered saline (PBS) for cell washing
Fixing solution
Fix/perm solution
Primary antibodies (anti-COX-2, anti-iNOS, anti-HO-1, anti-SOD, anti-nitrotyrosine)
Secondary antibody
Fluorescence-activated cell sorter (FACSscan)
Cell Quest software (version 4)

positive controls

Not specified

negative controls

Not specified

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