Multifunction microplate reader

With the purpose of evaluating colon permeability, serum levels of fluorescein isothiocyanate (FITC)-Dextran 4000 (FD-4) were determined. Mice were fasted for 20 h and then orally gavaged with FD-4 (60 mg/100 g). After 4 h, blood samples were collected and a multifunction microplate reader (BioTek Instruments, Inc., USA) was used to detect the level of FD-40 in serum in black 96-well plates at 490 nm of excitation wavelength and 520 nm of emission wavelength.

When the cells reached 70–80% confluence, 1 × 10⁵ cells were seeded in 24-well plates. To verify the relationship between lncRNA TCONS_00007391 and CD86, each recombinant plasmid (800 ng) was co-transfected with internal vector pRL-TK (20 ng) using Lipofectamine™ 8000 reagent according to the manufacturer’s protocol. After 48 h post-transfection, the luciferase activity was detected using the dual luciferase reporter assay system (Vazyme, Nanjing, China) and the pGL3-basic vector was used as a negative control. The firefly luciferase and Renilla luminescence activities were measured in a multi-function microplate reader (Biotek, Winooski, VT, USA).

MTT assay was applied to examine cytotoxicity of the candidates in HCT-116 cells. Cells seeded into 96-well plates at a density of 1 × 10³ cells/well were incubated for 24 h, then cells were treated with or without indicated concentrations of the candidates for 48 or 96 h. After incubation, 10 μL per well of MTT solution (5 mg/mL) was supplied and incubated for 4 h. After discarding the suspension, each well was added into 100 μL of DMSO, and the absorbance was determined by a multifunction microplate reader (Biotek., USA) at 570 nm. The assays were carried out in triplicate.

LX-2 cells were supplied by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Cell lines were cultured in DMEM, replenished with 10% (V/V) FBS and 100 U/mL penicillin/streptomycin, and grown in an incubator at 37 °C with 5% CO₂. LX-2 cells were counted after detachment with trypsin. A total of 1 × 10⁴ cells per well with or without 10 ng/mL TGF-β1 and/or PB (1, 2, or 4 μM) were added into a 96-well plate and cocultured for 24 h. After, 5 mg/mL of MTT was added to the wells, which were cultured for another 4 h. After removal of the suspension, the formazans were dissolved in DMSO and detected using a multifunction microplate reader (Biotek, Bad Friedrichshall, Germany) at a wavelength of 490 nm.

For infection of A549 cells, single colonies were expanded by resuspension in brain heart infusion broth and incubation at 37°C until the midlog phase (OD₆₀₀, 0.3–0.4), and then were harvested by centrifugation. A549 cells were inoculated with S. pneumoniae resuspended in cell culture medium without antibiotics at 37°C and 5% CO₂. For each experiment, a new aliquot of bacteria was slowly thawed and added to the cell medium at a multiplicity of infection of 20 (i.e. 20 pneumococci per epithelial cell). All experiments were performed in triplicate.
A549 cells were seeded at a density of 5 × 10⁴ cells per well (100 μL) in 96‐well plates. At 70–80% confluence, the cells were incubated with S. pneumoniae at 37°C for the indicated times. Cell viability was measured using Cell Counting Kit‐8 (Dojindo Molecular Technologies, Kumamoto, Japan), following the manufacturer’s instructions. The absorbance was measured in a multifunction microplate reader (BioTek, Winooski, VT, USA) at 450 nm.

The following instruments were used in this study: Microwave extractor (Xinyi Microwave Chemical Technology Co., Ltd. Shanghai, China), Sartorius BS214-D Balance (Sedris Scientific Instruments Co., Ltd., Beijing, China), KQ3200 ultrasonic cleaner (Kunshan Ultrasonic instrument Factory, Suzhou, China), Automatic triple water distiller (Yarong Biochemical Instrument Co., Ltd., Shanghai, China), Uv-1750 UV spectrophotometer (Shimazu Technology Co., Ltd., Beijing, China), Soxhlet extractor (Nanchang University Glass Instrument Factory, Nanchang, China), GT10-1 high-speed desktop centrifuge (Beijing Times Beili Centrifuge Co., Ltd., Beijing, China), Quanta 250 scanning electron microscope (Oregon, USA), Triple TOF5600 + mass spectrometer, Shimadzu Corporation (Jingdu, Japan), multi-function microplate reader from Biotek (Winooski, VT, USA), X-ray diffractometer (TD-3500 X-ray diffractometer, Shanghai, China), Thermogravimetric analyzer (TG/DTA6300, Seiko, Jingdu, Japan), FT-IR spectrometer (Spectrum Two Platinum spectrometer, Elmer, Waltham, MA, USA), ultra-clean workbench from purification company (Suzhou, China), and inverted microscope from Leica (Wetzlar, Germany).

DF-1 cells were seeded in 96-well plates and co-transfected with plasmid of wild-type 3′UTR or mutant 3′UTR with mimic or mimic-NC. After 48 h, the luciferase activity was detected using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA) following its instruction. The firefly luciferase and Renilla luminescence activities were detected using multi-function microplate reader (Biotek, Winooski, VT, USA).