Victortm x3

The enzymatic activity of urease from H. pylori was measured by a colorimetric assay, based on the conversion of urea to ammonia (NH₃), according to a previous article [34 (link)]. The production of NH₃ generates an increase in pH, detectable by the pH indicator phenol red (0.001 g/L), diluted in a phosphate buffer containing urea 2% w/v (9.1 g/L KH₂PO₄, 9.5 g/L Na₂HPO₄). Plant extracts were aliquoted in 96-well plates in the presence or absence of adherent GES-1 cells (seeded 10⁴ cells/well for 48 h). Then, 100 μL of bacterial suspension (OD = 0.1, PBS 1×) and 100 μL of 2% urea buffer were added. NaF (1 mM), known as a chelating agent of Ni²⁺, was used as a reference inhibitor [34 (link)]. The shift of absorbance to 570 nm was measured using a spectrophotometer after 1 h (Victor^TM X3, PerkinElmer, Waltham, MA, USA).

In order to explore the effect of let-7 miRNA on the level of oxidative stress, we carried out estimation of ROS levels using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay following the protocol as described by Kaur et al. (2012) (link). Worms of control and let-7 miRNA silenced groups were washed thrice with M9 buffer and twice with phosphate buffer saline (PBS). An approximate number of 100 worms/100 μl assay solution, were transferred to assay wells of OptiPlate-96 F (Perkin Elmer) with each group being assayed in triplicates. A volume of 100 μl H₂DCFDA (Cat. No. D399, Invitrogen) from an ethanol stock of 100 μM, was added to each well. Fluorescence from each well was quantified at three time points – (i) before addition of the dye, (ii) immediately after addition of the dye, and (iii) post 1 h incubation of addition of the dye. The Fluorescence intensity measurements were carried out using Multimode plate reader (Perkin Elmer, VICTOR^TM X3), at excitation wavelength 485 nm and an emission wavelength 520 nm. The change in fluorescence was calculated by subtracting initial reading from the final reading; the numbers were presented as fluorescence intensity per worm and plotted as mean ± SE. Statistical significance was calculated by student’s t-test using GraphPad Prism software package.

PSGL-1 peptide (ATEYEYLDYDFL) was coated on the amine-binding ELISA plate at 37 °C for 2 h in phosphate-buffered saline (PBS). Subsequently, to block the remnant amine binding sites, 3% milk was added to PBS at RT for 1 h. PTS on the immobilized PSGL-1 was catalyzed by the PST–TPST coupled enzyme system, followed by washing with 0.05% Tween-20/PBS thrice. The reaction mixture was incubated with primary (a mouse anti-sulfotyrosine antibody diluted with PBS, 1:1000) and secondary antibodies (an anti-mouse HRP-conjugated antibody, 1:5000) at RT for 1 h and 30 min, respectively. After each interval of the experimental procedure, the ELISA plate was washed with PBS/0.05% Tween-20 for three times. Finally, 100 μL of tetramethylbenzidine substrate for HRP was added and incubated at RT for 30 min. The HRP-catalyzed reaction was terminated using 100 μL of 2 M H₂SO₄, and the resulting chemiluminescence was quantified at OD450 nm by using an ELISA reader (VICTORTM X3; Perkin Elmer, Waltham, MA, USA).