Synergy hi microplate reader

Luciferase reporter plasmids containing SGT1 and TRAF3 were constructed (Sangon Biotech, Shanghai, China). The plasmids were extracted (D6950-01, Omega, Doraville, GA, USA), their concentration was determined, and the DNA was stored at −20°C for subsequent experiments. HEK293 cells were inoculated in a 24-well plate and grown to about 70% to 90% confluence before being co-transfected (L3000015, Invitrogen™, Carlsbad, CA, USA) with pcDNA3.1, HSF1-constitutive plasmids, luciferase reporter plasmids, PGL-basic, and pRL-TK. After 48 h of transfection, the cell lysate was extracted and tested using a dual-luciferase reporter assay kit (E1910, Promega, Madison, WI, USA) and loaded onto a luminometer for the measurement of luminescence (Synergy^HI Microplate Reader, BioTek, Winooski, VT, USA).

The Cellular Reactive Oxygen Species Detection Assay Kit (AbCam) was used according to the manufacturer’s instructions. Cells were seeded and stimulated for 72 hr before the assay was carried out with 25 μM DCFDA. Samples were analyzed on a Synergy HI microplate reader (BioTek). Results were first normalized to a positive and negative control.

On day 150, sediments in each jar were homogenized, and then sub-samples were taken, immediately frozen and then stored at -20°C for microbial community analysis. Each treatment quadruplicate sample was thawed, and DNA was extracted in duplicate using the MoBio PowerSoil kit (MoBio, Carlsbad, CA, United States). Quadruplicates from each treatment were paired, and had their DNA extracts combined, resulting in duplicate DNA samples representing each treatment, i.e., four replicates of the TYP 10% was reduced to two for sequencing (each constituting four DNA extractions from two jars). Pooling DNA was done to reduce bias of small material amounts that makes up one DNA extraction, and to increase DNA concentrations for downstream work. DNA was concentrated down into 40 μL aliquots suspended in the C6 solution from the MoBio PowerSoil kit. Sample DNA was quantified using a Take3 spectrophotometry system on a Synergy HI microplate reader (BioTek, Winooski, VT, United States).

The RSV-specific IgG antibodies and the antibody subtypes (IgG1 and IgG2a) were analyzed using an enzyme-linked immunosorbent assay (ELISA) using F-protein (BEI Resources) as the coating antigen [26 (link),28 (link),29 (link),30 (link)]. Briefly, 96-well microplates (Nunc, Rochester, NY, USA) were coated with F-protein (200 ng/well) and incubated overnight at 4 °C. The plates were then washed with PBST (PBS with 0.05% Tween 20) and followed by a blocking step with 3% BSA for 90 min at 37 °C. The serum or lung homogenates were serially diluted with PBS and added to the wells followed by incubation for 90 min at 37 °C. Next, the wells were washed thrice with PBST, after which the secondary antibody conjugated to horseradish peroxidase (HRP) IgG, IgG1, IgG2a (serum and lung), and IgA (lung) (Invitrogen, Waltham, MA, USA), at a 1:2000–4000-fold dilution, was added to the wells. After incubation for 90 min at 37 °C, the plate was washed three times with PBST followed by the addition of the substrate tetramethybenzidine (TMB) (BD Bioscience, San Jose, CA, USA). The color reaction was stopped using 0.3 M sulfuric acid after incubating the TMB for 15 min. The optical density (OD) at 450 nm was measured using a Synergy HI microplate reader (BioTek, Winooski, VT, USA).