Spectramax m5 reader

For the determination of ATP production the CellTiter-Glo Assay (CTG) and for the determination of dehydrogenase activity the CellTiter-Blue Assay (CTB) were employed (all from Promega, Mannheim, Germany) according to the instructions of the manufacturer, and as described previously [27 (link)]. All measurements of luminescence and fluorescence were performed using either a Mithras LB 940 Multimode Microplate reader (Berthold Technologies, Bad Wildbad, Germany) or a Spectra Max M5 reader (Molecular Devices, Biberach, Germany).

The S. suis strains used in the present study are listed in Table 1. S. suis strain S10 is a virulent isolate from an infected pig, and its genome is 99% identical to the genome of S. suis 2 strain P1/7 (de Greeff et al., 2011 (link)), a sequenced reference strain of which the genome had been annotated previously (Holden et al., 2009 (link)). S. suis was grown at 37°C at 5% atmospheric CO₂ in Todd Hewitt Broth (THB, Thermo Scientific, Oxoid) or on THB plates containing 1.2% of agar (BD). When required the medium was supplemented with spectinomycin (Invitrogen) and/or chloramphenicol (Sigma) at a concentration of 100 and 5 μg/ml, respectively. Insertional deletion mutants of the genes cinA, oppA, and comYC were constructed in S. suis strain S10 by Gene Splicing Overlap Extension PCR (SOE-PCR) and allelic replacement as previously described (Zaccaria et al., 2014 (link)). The primers used for SOE-PCR are shown in Table 1. Successful deletion of the genes was verified by colony PCR using primer combinations based on DNA sequences of the inserted DNA and proximal chromosomal DNA (Table 1) and verified by sequencing of the amplicons. Growth phase was determined by measuring optical density at 600 nm (OD_600nm) using a SpectraMax M5 reader (Molecular Devices LLC).

An aggregation assay was conducted according to the method reported in a previous study [26 (link)]. Briefly, the final concentration of pathogens and probiotics were adjusted to 2 × 10⁸ CFU/mL. For auto-aggregation assay, the resuspended probiotics (5 mL) were added into sterile tubes separately and then placed at 37 °C without agitation. Then, an aliquot of 150 μL upper suspension was taken at 3 h intervals to measure the absorbance of OD_600nm using a SpectraMax M5 reader (Molecular Devices, Sunnyvale, CA, USA). Finally, the auto-aggregation rate was calculated by the equation: auto-aggregation rate (%) = (A0 − An)/T0 × 100%, where A0 is the OD_600nm value of upper suspension at 0h, and An is the OD_600nm value at different time points.
For the co-aggregation assay, equal volumes (1.5 mL) of lactic acid bacteria and pathogens cultures at logarithmic growth phase were mixed completely and placed at 37 °C without agitation. Then, an aliquot of 150 μL upper suspension was taken at 0 h and 4 h to measure the absorbance of OD_600nm using a SpectraMax M5 reader. Finally, the co-aggregation rate was calculated by the equation: co-aggregation rate (%) = [(A_pro + A_pat) − 2 × A_mix]/(A_pro + A_pat) × 100%, where A_pro and A_pat are the OD_600nm value of lactic acid bacteria and pathogen cultures at 0 h, and A_mix is the OD_600nm value of the mixed resuspensions at 4 h.

The PROM ELISA was as follows: 96-well streptavidin-coated ELISA plates (cat. no. 655995, Greiner Bio-One, Austria) were coated with 2.5 ng/mL biotinylated peptide dissolved in assay buffer (10 mM PBS-BTB, 8 g. NaCl, pH 7.4), 100 µL/well and incubated for 30 min at 20 °C in the dark with 300 rpm shaking. Plates were washed five times in washing buffer (20 mM TRIS, 50 mM NaCl, pH 7.2). Subsequently, 20 µL of selection peptide or sample were added to appropriate wells, followed by 100 µL of 75 ng/mL horseradish peroxidase (HRP) conjugated monoclonal antibody. The plates were incubated for 20 h at 4 °C with shaking, and subsequently washed in washing buffer. Hundred µL per well of BM Chemiluminescence ELISA Substrate (POD) (cat. no. 11582950001, Roche, Switzerland) working solution was then added to the plate and incubated for 3 min at 20 °C with shaking. The plate was analyzed by a SpectraMax M5 reader (Molecular Devices, CA, USA) with settings: luminescence, Lm1 = 440 nm, Lm2 = 650 nm. A standard curve was generated by serial dilution of the selection peptide and plotted using a 4-parametric mathematical fit model. Standard concentrations were 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016 and 0 ng/ml. Each plate included five kit controls to monitor inter-assay variation. All samples were measured within the measurement range of the assay.

The experiment was performed as described [23 (link)]. NF-κB promoter region was cloned into pGL3-based vectors to construct Luciferase reporter plasmid. The reporter plasmid was transfected into RA-FLS-Ctrl or RA-FLS-TRIP groups using the Lipofectamine 2000 (Invitrogen, Shanghai, China), and phRL-TK plasmid was cotransfected as internal control. 36 hours later, the Luciferase activities were measured on a SpectraMax M5 reader (Molecular Devices, California, USA) using the Dual-Luciferase Reporter Assay System (Promega, Madison, USA).

Cell proliferation after transfection with hsa_circ_0061276 overexpressing plasmids or hsa_circ_0061276 shRNA plasmids (Geneseed) was detected using Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan). After culturing for 24 h, 48 h, 72 h, and 96 h, 10 μl/well of CCK-8 reagent was added to the cultures in the dark, and then the cultures were incubated for 3 h. The SpectraMax M5 reader (Molecular Devices, Silicon Valley, CA, USA) was used to detect the plate optical density (OD) at 450 nm.
Forty-eight hours post-transfection with hsa_circ_0061276 overexpressing plasmids or shRNA plasmids (Geneseed), colony formation experiments were performed as previously reported (19 (link)). Forty-eight hours after cell transfection, cells were inoculated into a six-well plate (1,000 cells per well). After culturing cells for 10 days, colonies were first stained with crystal violet (Solarbio, Beijing, China) and then counted using Photoshop software (Adobe, San Jose, CA, USA).