Pge2 elisa kit

HaCaT cells (5 × 10⁴ cells/well) were seeded in 24-well plates in 0.4 ml DMEM, and allowed to adhere overnight. The cells were washed and incubated with FCS-free DMEM for 24 hours. The culture was incubated in the presence of vehicle (0.07% DMSO) or indicated concentrations of VRCZ for 6 hours. The supernatant PGE₂ amounts were quantified using a PGE₂ ELISA kit (highly sensitive kit for inflammation and eicosanoid research, ADI-900-001; Enzo Life Sciences, Farmingdale, NY) according to the manufacturer’s protocol.

Cytokine and chemokine levels in kidneys of mice with systemic C. albicans infection were measured by digesting the excised organ in 1 mL LiberaseTL and DNAse I solution at 37°C for 20 minutes, followed by straining through a 40 mm filter, and centrifugation at 12,000g for 45 minutes at 4°C. Supernatants were collected and stored at –80°C. Cytokine/chemokine analysis of these samples was performed at EVE Technologies using the Mouse Cytokine Array/Chemokine Array 44-Plex (MD44) immunoassay. PGE₂ levels in kidney supernatants were measured using the PGE₂ ELISA kit (Enzo Lifesciences, catalog ADI-900-001). Plates for PGE₂ were read at 405 nm using a Varioskan instrument (Thermo Fisher Scientific), as we previously reported (8 (link)).

To determine the anti-inflammatory activity of 1.0, 10.0, or 100 μg/mL PLL-EO, an enzymatic cell-free in vitro assay using human recombinant cyclooxygenase (COX-1, COX-2) (Sigma-Aldrich; Prague, Czech Republic) was performed [27 (link)]. The activity was expressed as the percentage of cyclooxygenase inhibition by PLL-EO compared to untreated samples (0.0 μg/mL PLL-EO). Briefly, COX-1 (1 unit/reaction) or COX-2 (0.2 unit/reaction) was added to 180 μL of the incubation mixture (100 mM Tris buffer pH 8.0; 5 μM porcine hematin; 18 mM L-epinephrine; 50 μM Na₂EDTA) in a 96-well plate. PLL-EO (1.0, 10.0, and 100 μg/mL) was dissolved in ethanol (10 μL) and the reaction was started with 10 μM arachidonic acid. Ibuprofen (1.0, 10.0, or 100 μg/mL) was used as the positive control. The reaction was stopped after 20 min by adding formic acid (10%). The main product of this reaction, PGE₂, was quantified using a PGE₂ ELISA kit according to the manufacturer’s instructions (Enzo Life Sciences, New York, NY, USA). The absorbance was recorded at 405 nm (Tecan Infinite M200 microplate reader). Experiments were repeated three times with three technical replicates.

4-Octyl itaconate was initially supplied by Professor Richard Hartley and results were later confirmed with commercially available 4-OI (Sigma Aldrich). Pam3CSK4, dimethyl fumarate, diethyl maleate, indomethacin and NS-398 (Sigma Aldrich) were also used. Antibodies used were anti-β-actin (Sigma Aldrich), anti-COX2 (Abcam), anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-NRF2, anti-KEAP1, anti-ATF4, anti-phospho-NF-kB p65 (Ser536), anti-NF-kB p65, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK, anti-phospho p44/42 MAPK (Erk1/2) (Thr202/Tyr 204) and anti-p44/42 MAPK (Erk1/2) (Cell Signaling). Anti-mouse IgG and anti-rabbit IgG secondary horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch) were also used. A PGE₂ ELISA kit was used (Enzo Life Sciences). The Silencer Select siRNAs against NRF2 (s70522), ATF4 (s62689) and Anxa1 (s69299), as well as the Silencer Select negative control, were used (ThermoFisher Scientific).

Following siRNA knockdown, to determine the levels of molecules exported from cells, ELISAs were carried out on the media. As a control, 3 wells of nontransfected cells were treated with 50-µM MK571. For cAMP, cells were first stimulated with 100-µM forskolin for 2 h at 37 °C. For PGE₂, cells were stimulated with 10-µg/mL lipopolysaccharides (LPS) for 24 h at 37 °C and with 80-nM phorbol 12-myristate 13-acetate (PMA) for 1 h at 37 °C, respectively. For S1P, there was no stimulation. The cell culture medium was then replaced with a fresh cell culture medium, and the cells were incubated for 18 h at 37 °C. The supernatants (conditioned media) were centrifuged at 1000× g to pellet out any floating cells and collected and stored at −80 °C or used immediately for determination of cAMP by using the cAMP ELISA kit (Enzo Life Sciences Inc., Exeter, UK), for the determination of PGE₂, using the PGE₂ ELISA kit (Enzo Life Sciences Inc.), or for S1P, using the human S1P ELISA kit (Abbexa Ltd., Cambridge, UK), according to the manufacturer’s instructions. The cells were also harvested for protein quantification.

The assay was performed according to the procedure previously described by Reininger and Bauer [15 (link)] with COX-1 from ram seminal vesicles and human recombinant COX-2. COX-1 (1 unit/reaction) or COX-2 (0.5 unit/reaction) was added to 180 μL of incubation mixture that consisted of 100 mM tris buffer (pH 8.0), 5 μM porcine hematin, 18 mM L-epinephrine, and 50 μM Na₂EDTA. The wine sample, tested compound diluted in DMSO, 12% ethanol (in case of blanks for the wine samples), or pure DMSO (in case of blanks for purified constituents) was added (10 μL) and the mixture was preincubated for 5 min at room temperature. The addition of 5 μL of 10 μM AA started the reaction. After 20 minutes of incubation at 37°C, the reaction was stopped by 10 μL of 10% formic acid. All samples were diluted 1 : 15 in ELISA buffer and the concentration of (prostaglandin E₂) PGE₂ produced by the reaction was determined by a PGE₂ ELISA kit (Enzo Life Sciences, US) according to the manufacturer's instructions. Absorbance relative to PGE₂ concentration was measured with a microplate reader Tecan Infinite M200 (Tecan Group, Switzerland) at 405 nm. The results were expressed as percentage inhibition of PGE₂ formation against untreated samples (blanks).