Black 96 well plate

Neutrophils (5 × 10⁴ cells/well) from healthy controls and MIPP patients were incubated with A. actinomycetemcomitans or JP2 at a MOI-10 ratio in a 96-well black plate (PerkinElmer, Waltham, MA). HOCl (0.0006%) served as positive control. DCFH-DA (7′-dichlorofluorescein diacetate, Sigma–Aldrich, Israel) was added to each well as a marker for ROS. Immediately after addition of the bacteria, the plate was transferred to a florescence plate reader (infinite plate reader; Tecan, Männedorf, Switzerland) at 37˚C with readings (490nm excitation and 535nm emission) every 5 min for a total 150min.
The experiments were repeated in triplicate for 8 MIPP patients and 8 healthy controls.

293T cells that stably expressed human ACE2 (293T-hACE2) and lentiviral-based pseudotyped SARS-CoV-2 viruses were provided by National RNAi Core Facility (Academia Sinica, Taiwan). One day before neutralization assay, 293T-hACE2 cells were seeded into 96-well black plate (Perkin Elmer, MA, USA) at a density of 1 × 10⁴ cells per well at 37 °C. Mouse sera were inactivated at 56 °C for 30 min and then serially diluted by four folds with culture medium before incubation with indicated SARS-CoV-2 pseudovirus for an hour. The mixtures were then added to pre-seeded 293T-hACE2 cells and incubated for 3 days. Luciferase activity was measured by Luciferase Assay kit (Promega, WI, USA). The 50% neutralization titer (NT₅₀) was calculated by nonlinear regression using Prism software version 8.1.0 (GraphPad Software Inc.).

Vero-E6 GFP cells were seeded at 10⁴ cells/well in 96-well black plates (PerkinElmer). The following day, cells were incubated, with or without compounds, with 2 μM P-gp inhibitor CP-100356 [47 (link)]. The media was removed 24 h after treatment, and the total well GFP fluorescence was measured using Victor NIVO 5 with 485/535 nm excitation wavelength. The cytotoxic effect of compounds was calculated as a percentage of cell viability with respect to untreated control. Data were analyzed using Prism 9. Version 9.1.2.

293 T cells that stably expressed human ACE2 (293T-hACE2) and lentiviral-based pseudotyped SARS-CoV-2 viruses were provided by the National RNAi Core Facility (Academia Sinica, Taipei, Taiwan). One day before neutralization assay, 293T-hACE2 cells were seeded into 96-well black plates (Perkin Elmer, MA, USA) at 1 × 10⁴ cells per well at 37°C. Mouse serum was inactivated at 56°C for 30 min and serially diluted by 4-fold with culture medium before incubation with the indicated SARS-CoV-2 pseudovirus for 1 h. The mixtures were then added to pre-seeded 293T-hACE2 cells and incubated for 3 days. Luciferase activity was measured using the Luciferase Assay kit (Promega, WI, USA). The 50% neutralization titer (NT₅₀) was calculated using nonlinear regression with Prism software 8.1.0 (GraphPad Software Inc.).

The inhibitory effect of the oligopeptide against SrtA and its mutants activity was determined as previously described in 96-well black plates (PerkinElmer, Boston, MA, United States) (Wang et al., 2015c (link)). Briefly, 100 μL of purified SrtA (4 mM) was preincubated with 100 μL of increasing concentrations of the oligopeptide (3.125–25 μM) in reaction buffer at 37°C for 20 min. The model fluorescent peptide substrate Dabcyl-QALPTTGEE-Edans (GL Biochem, Shanghai, China) (final concentration of 10 μM) was added to the reaction system, followed by incubation at 37°C for 1 h. Fluorescence intensity was measured at emission and excitation wavelengths of 350 and 520 nm, respectively. The experiments were repeated three times independently for each sample.

Nitrites were measured in culture medium supernatants after 24 h of incubation with the compounds. The fluorometric assay of nitrite is based on the reaction of nitrite with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole. A 200 µM working nitrite standard was prepared from a 2.0 mM sodium nitrite stock solution in endotoxin-free deionized water (Sigma-Aldrich). A working DAN solution of 50 µg ml⁻¹ was prepared by diluting a 20 mg ml⁻¹ stock solution with 0.62 M HCl. All assays were conducted in 96-well black plates (Perkin Elmer). In each well, 20 µl of standard or 30 µl sample, were added, respectively, to 80 or 70 µl of endotoxin-free deionized water. Then, 10 µl of working DAN solution was added to each well and the plates were shaken. The plates were incubated at 23 °C for 10 min. After then, 20 µl of 2.8 M NaOH was added to each well, and the plates were shaken again. Then the plate was incubated in the dark for 1 min and measured in a fluorescence plate reader (POLARstar Omega, BMG Labtech) with an excitation of 355 nm and an emission of 460 nm. The results were expressed as percent of control sample (the solvent).