1420 multilabel counter victor3 5

Mouth coating (QRB_%), defined as the residual food that sticks to the oral surface after food ingestion, was quantified by the “mouth rinse” method [26] (link). The lipids of the residual food were quantified by the intensity of curcumin fluorescence in the rinse water.
The quantity of curcumin (Naturex, France), used as a food colour (E100), was added during cheese production to reach a level of 30 mg.kg⁻¹ in the final cheeses. To measure the amount of cheese remaining in the mouth, each subject was asked to place a piece of cheese (6 g, at 17°C) in the mouth and to chew normally until they needed to swallow. The subjects swallowed without cleaning movement and then rinsed their mouth (with cleaning movements) with 4 mL of warm water at 50°C for 30 s, and spat it into a vial. This rinsing procedure was applied two times consecutively and the spittle was cumulated in the same vials. The fluorescence intensity of curcumin was quantified using a Perkin Elmer 1420 Multilabel Counter Victor 3 V at an excitation wavelength of 450 nm and an emission wavelength of 510 nm. All measurements were done in triplicate.

Fluorescence intensity was measured with a 1420 Multilabel Counter (Victor3 V; Perkin-Elmer, Waltham, MA). The cell density was measured at 600 nm (Eppendorf BioPhotometer, Germany), and the excitation and emission wavelengths for green fluorescent protein (GFP) were 485 ± 20 and 585 ± 20 nm, respectively. The fluorescence intensity (a.u.) was normalized to the cell density (OD₆₀₀). To measure the fluorescence of the strains with different malonyl-CoA levels, an overnight culture was collected, inoculated into fresh SC medium with an initial OD₆₀₀ of 0.2 and cultivated for 12 h. The fluorescence intensity was then measured. For mutant strains screening, strains were picked from resistance medium plate and inoculated into 200 μL of fresh SC medium in 96-well plates. The OD₆₀₀ and fluorescence were detected using a Multi-Detection microplate reader (Synergy HT; BioTtek, Winooski, VT).

Stable transfected cells were seeded onto 96-well microliter plates and cell proliferation was measured with the cell proliferation Reagent WST-1 (5015944001; Sigma-Aldrich) according to the manufacturer’s recommendations. The absorption of WST-1 was measured at 450 nm using a Multilabel counter 1420 Victor³ V (Perkin Elmer, Wellesley, MA, USA).

To assess Rac1 activation Rac1 G-LISA kit (Cytoskeleton) was used according to the manufacturer’s protocol. Cells were cultured first for 12 h in minimal essential medium (MEM) (Gibco) containing 5% fetal bovine serum (FBS, Thermo Fisher Scientific) after which they were washed with PBS and cultured for an additional 12 h in MEM without FBS. Then cells were washed with PBS and cultured for an additional 4 h in MEM without FBS with or without adding Rac1 inhibitor ZINC69391 to final concentration of 100 µM. Then cells were washed with PBS and incubated for 5 min with EGF (Peprotech) at concentration 100 ng/ml. Protein was isolated using standard G-LISA buffer GL36 (Tris pH 7.5, MgCl₂, NaCl, IGEPAL and SDS). Obtained lysate was aliquoted and frozen in liquid nitrogen. The Rac1 G-LISA kit (Cytoskeleton) contains a Rac-GTP-binding protein linked to the wells of a 96 well plate. Active, GTP-bound Rac1 in cell/tissue lysates will bind to the wells while inactive GDP-bound Rac1 is removed during washing steps. The bound active Rac1 is detected with a Rac1 specific antibody. The degree of Rac1 activation is determined by comparing readings from activated lysates versus non-activated lysates. Plates were read in a Multilabel Counter 1420 VICTOR3V (Perkin Elmer, Waltham, Massachusetts, USA) at 450 nm.