Synergy lx

The FITC dextran assay was conducted as previously described [41 (link),42 (link)]. Briefly, food and water were removed from mice for 4 h followed by oral gavage with 500 mg/kg FITC dextran (Sigma Aldrich, St. Louis, MO, USA) reconstituted in sterile PBS. Mice were euthanized and blood collected via cardiac puncture. The FITC dextran levels in the serum were measured using a fluorimeter (Agilent BioTek Synergy LX, Santa Clara, CA, USA), and the concentration was calculated using an FITC standard curve.

Cells were cultured identically to previous, except they were plated in 96 well cell culture treated plates and exposed to gamma-irradiated Mtb for a total of 24 h to facilitate VEGF production and secretion. Supernatants were collected and spun down and then the upper layer was collected for further analysis. ELISA was performed according to the manufacturer’s instructions (R&D Systems #DY293B). Absorbance was read on an Agilent Synergy LX plate reader.

HEK-293 T cells were seeded into a 96-well plate and allowed to reach 50–70% confluence prior to transfection. The pmiR-RB-Report™ luciferase vector (RiboBio, China) was modified by the introduction of either wild-type or mutant TET2 fragments (524 bp) into its restriction sites and designated as LUC-WT-TET2 or LUC-MUT-TET2, respectively. The transfection was performed using lipofectamine 3000 (Invitrogen, USA) with 150 ng of plasmids of LUC-WT-TET2 and LUC-MUT-TET2, and then exposed to 100 nM of m-NC and miR-20b-5p mimic, 300 nM of i-NC and miR-20b-5p inhibitor. The culture medium was replaced 6 h post-transfection. After 48-h incubation, the Dual-Luciferase system (Promega, USA) was utilized to determine the firefly and Renilla luciferase activities. The medium was aspirated, and the cells were treated with 50 μL of PBS and luciferase reagent. The 96-well plate was then incubated on a shaker at room temperature for 10 min before the determination of firefly luciferase activity. The Renilla luciferase activity was determined by adding 30 μL of stop reagent per well and shaking for 10 min. The luciferase activities were then detected using Agilent BioTek Synergy LX. Finally, the differences between firefly and Renilla luciferase activities were calculated to determine the relative luciferase activity.

Cell viability was assessed using CellTiter-Glo (Promega, Madison, Wisconsin, USA). Briefly, 1000 cells were seeded in white, flat-bottom, 96-well plates (Corning, Corning, NY, USA). After 24 h, drugs were added to the medium, and cells were incubated for 72 h. CellTiter-Glo luminescent reagent was added according to the manufacturers protocol, and the luminescence signal measured on a Synergy LX (Agilent, California, USA) with BioTek Gen5 (v3.08). To evaluate if a combination of drugs is synergistic, cells were simultaneously treated with varying concentrations of drugs, and cell viability was measured with CellTiter-Glo. Synergism scores were obtained using the R package SynergyFinder (v2.2.4)^{75 (link)}.

Preparation of NGS libraries and sequencing were conducted by Seibutsu Giken Inc (Japan). The libraries were subjected to multiplexed deep sequencing in 200 bp paired-end mode on the NGS using a BGISEQ-G400 platform (MGI Tech, China). The concentration of the NGS samples was measured using either Synergy LX (Agilent Technologies, Santa Clara, CA) and QuantiFluor dsDNA System (Promega, Madison, WI), or Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and dsDNA HS Assay Kit (Thermo Fisher Scientific). The libraries for DNBSeq were prepared using either MGIEasy FS DNA Library Prep Set (MGI Tech) (25 ng of sample, 8 cycles) or MGIEasy PCR-Free DNA Library Prep Set (MGI Tech) (200 ng of sample, enzymatic digestion for 4 min). The quality of the prepared libraries was assessed using an Agilent 2100 bioanalyzer and a High Sensitivity DNA kit (Agilent Technologies), or Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies). The DNA nanoball was generated from the libraries using the MGIEasy Circularization Kit (MGI Tech).

The colorimetric CBA was performed as in our previous work [25 (link)]. Neuro-2a cells were trypsinised and suspended in culture medium (the same as for maintenance but with 5% FBS instead of 10% FBS). Then, Neuro-2a cells were seeded in a 96-well microplate at a density of 35,000 cells/well in 200 µL of culture medium for 24 h at 37 °C in 5% CO₂ humid atmosphere. Prior to exposure to TTX standard solution or pufferfish tissue extracts, some Neuro-2a cells were pre-treated with 20 µL of an ouabain and veratridine mixture in PBS at final concentrations of 0.125 and 0.2 mM, respectively. Tetrodotoxin standard solution and pufferfish tissue extracts were dried under an N₂ stream at 40 °C using a TurboVap evaporator (Zymark corp., Hopkinton, MA, USA), reconstituted in culture medium and serially diluted, and 10 µL was added to the wells with and without ouabain/veratridine pre-treatment. After 24 h, cell viability was measured using the MTT assay (Manger et al. 1993). Absorbance at 570 nm was measured with a Synergy LX microplate reader from BioTek (Agilent Technologies, Inc., Santa Clara, CA, USA). Measurements were performed in triplicate.