Microtiter plate reader

For the MTT assay, 5×10³ cells per well were seeded with complete growth medium on a 96-well plate. The cells were divided into three groups: miR-93-5p NC group, miR-93-5p mimic group and miR-93-5p inhibitor group. Treated cells were assessed for 1 to 3 days via the previously described MTT assay. The data were measured using a microtiter plate reader with a 570 nm filter (Thermo Scientific).

The activity of caspase-3 was detected by the caspase-3 colorimetric assay kit. Briefly, 6 h after the last injection, the young rats were sacrificed by decapitation, and the brain tissue was quickly separated. Fresh frozen brain tissue was homogenized in lytic buffer to extract protein or extract protein directly from PC12 neural cell line. The protein concentration was then determined using the Bradford Protein Assay Kit. After that, 50 μL of the lysate supernatant containing 200 μg of protein, 50 μL of reaction buffer, and 5 μL of caspase-3 substrate was incubated in a 96-well plate at 37°C for 4 h with a microtiter plate reader (ThermoFisher, Shanghai, China). The absorbance was measured at a wavelength of 400 nm. The absorbance of the blank sample without the caspase-3 substrate was subtracted from these values. The results implied that the final caspase-3 activity was the ratio of the control group.

MTT was converted to formazan crystals using mitochondrial dehydrogenase, and cell viability were determined by MTT assay. According to the experimental design, the cells supplemented with 10 μL of 5 mg/mL MTT solution was incubated at 37°C for 4 h. The medium containing MTT was removed, and 200 mL of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystals. The absorbance was measured at 492 nm using a microtiter plate reader (ThermoFisher, Shanghai, China). The absorbance of the control group (A control) was set to a survival rate of 100%. The absorbance of the treated cells (A experimental) correlated with the absorbance of the control cells and was normalized. The background was the absorption rate of the medium plus MTT in the absence of cells. Cell survival rate was defined as cell viability = [(A experimental − A background)/(A control − A background)] × 100%.

We followed the methods of Lai et al. [26 (link)]. The radio immunoprecipitation assay (RIPA) lysis buffer (KeyGEN Biotech, Nanjing, China) which contained 0.1% protease inhibitor cocktail (KeyGEN) was used to extract total protein from BMSCs. The concentration of proteins was determined using a BCA kit according to the manufacturer's instructions (ATGene, Chongqing, China) and a microtiter plate reader (Thermo Fisher Scientific). 5 × SDS-PAGE sample buffer was added to equal amounts of protein, which were boiled at 100°C in a water bath for 10 min before being subjected to 8% SDS-PAGE.
Western blotting was performed as previously described [27 (link)]. Some adjustments were made, we used anti-RARG (Abcam, Cambridge, UK) (1 : 1000), anti-FRA1 (Abcam) (1 : 100), anti-CEBPA (Abcam) (1 : 1000), and anti-PPARG2 (Abcam) (1 : 500) antibodies according to the manufacturer's instructions (n = 6 per group). An ECL Prime kit (Millipore Corp, Billerica, MA, USA) and an ECL Imaging system (Syngene G : BOX, Cambridge, UK) were used to analyze the levels of immunoreactive proteins. ImageJ software was used to analysis the intensity of the protein bands.

The titer of PCV2d-capsid-specific IgG in serum collected from each mouse was measured using a commercial ELISA kit according to the manufacturer’s instructions (Meimian Biotechnology Co., Ltd., Yancheng, China). Briefly, microtiter plates were pre-coated with the PCV2 antigen. The mice serum was diluted 1:5 prior to incubation with the antigen. The HRP-anti-mouse IgG conjugate reagent was added to each well and incubated for 60 min at 37 °C. After washing, the chromogen solutions were added to each well for visualization. The optical density was measured using a microtiter plate reader (Thermo Fisher Scientific Inc., USA) at 450 nm.