Varioscan flash

Bioactive TGF-β was measured in the supernatants of liver pericyte cultures using transgenic Mink Lung Epithelial cells (MLECs), which express luciferase, downstream of the (PAI-1) promoter, corresponding to the expression of bioactive TGF-β (Abe et al., 1994 (link)). The assay was carried out as described in (Wipff et al., 2007 (link)). In brief, MLECs were plated as 25,000/ well and allowed to adhere for 3 hours following which their supernatant was replaced with either TGF-β standards, or the pericyte supernatant from control or treated wells. Measurement of total TGF-β was achieved by heat activation of the samples. After 20 hours of incubation, the MLECs were lysed and were read for luciferase activity using the Varioscan Flash (Thermo Scientific). Data were represented as a percentage of the total TGF-β in the respective sample.

Pre-cultures of P. tricornutum and S. marinoi grown under standard conditions in enriched f/2 media to mid-exponential growth phase were used to inoculate experimental cultures in fresh enriched f/2 with different concentrations of zeocin (InVivoGen, USA) in 48-well plates (Corning, USA). Growth was measured daily for one week from chlorophyll a fluorescence measurements (425 nm excitation and 680 nm emission wavelengths)¹⁷ using a plate reader (Varioscan Flash, ThermoScientific, USA).

The promoter assay was performed with the eGFP reporter plasmid pMEXGFP as previously described with the following modifications32 (link). eGFP expression was measured with a fluorometer (Varioscan flash, Thermo Scientific) with emission at 488 nm and excitation at 509 nm, and was normalized to cell growth (OD₆₀₀).

RV samples were homogenized on ice in a buffer containing, in mM, 5 HEPES, 1 EDTA, 5 MgCl₂, and 0.1% Triton-X100 [75 (link)]. Protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche, Bazel, Switzerland) and phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, Waltham, MA, USA) were added to the buffer immediately before use. CK activity in samples was measured spectrophotometrically from the production of NADPH, according to the Rosalki assay [76 (link)], with absorption measured at 340 nm. Next, β-mercaptoethanol (1 mM) was added prior to the CK activity assay, to prevent the oxidation of CK. Citrate synthase activity was measured using a spectrophotometer (Varioscan Flash, Thermo Scientific, Waltham, MA, USA) according to the enzyme assay described by Srere [77 ]. Enzyme activity was expressed in international units per mg protein (IU/mg).

AGE-derived fluorescence was measured as reported previously [21 (link)]. Briefly, 200 μL of the reaction mixture was used to measure fluorescence at an excitation wavelength of 370 nm and an emission wavelength of 440 nm by a Varioscan^® Flash (Thermo Scientific, Waltham, MA, USA) microplate reader. The value was calculated using the equation below.

Genomic DNA was isolated using DNeasy Plant Mini Kit (QUIAGEN) following the manufacturer’s protocol. The DNA integrity was examined by gel electrophoresis on 1.0% (w/v) agarose and the DNA amount was assessed by the spectrophotometry method.
The total DNA methylation was determined by the MethylFlash™ Methylated DNA Quantification Kit (Epigentek), which included a complete set of optimized buffers and reagents for colorimetric quantification of the global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC) in an ELISA-like microplate-based format (Varioscan Flash, Thermo Scientific).

Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and left to recover for 24 h. Then the cells were transfected with TGFβR2 siRNA or pCMV-TGFβR2 or their controls using Lipofectamine 2000 as the manufacture’s instruction. After the cells were cultured at 37°C with 5% CO₂ for 1–5 days, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide solution(MTT) (5 mg/ml in PBS) was added to each well and the plates were incubated for another 4 h. The formazan products of MTT were dissolved and the absorbance was measured at 490 nm using microplate reader (Varioscan Flash; Thermo Fisher Scientific). Experiments were performed in duplicate.