Multiskan fc microplate photometer

Plasma aspartate aminotransferase and gamma-glutamyl transferase catalytic activity were determined with a commercial kit from Biosystems S.A., following manufacturer instructions as previously described [13 (link)] using a Varioskan^TM Flash Multimode Reader (Thermo Fisher Scientific).
Quantification of free liver glucose and glycogen was performed in liver biopsies homogenized in 500 μL 2N HCl as previously described [8 (link)]. Briefly, homogenates were subjected to 100°C during an hour for glycogen digestion to glucose by acid-heat hydrolysis [24 ]. Free liver glucose and digested liver glycogen were determined using a kit from Biosystems S.A., following manufacturer instructions after neutralizing acid samples with an equal amount of 2M NaOH. Absorbance was measured at λ = 505 nm using a Multiskan^TM FC Microplate Photometer (Thermo Fisher Scientific).
For liver triglyceride quantification, liver homogenates were performed according to Armour et al. [25 (link)]. In brief, liver biopsies were homogenized in lysis buffer (140 mM NaCl, 50 mM Tris and 1% Triton X-100, pH 8) and measured using a kit from Biosystems S.A., following manufacturer instructions at λ = 505 nm using a Multiskan^TM FC Microplate Photometer (Thermo Fisher Scientific). For all metabolite assays intra and inter-assay coefficient of variation (CV) were less than 10%.

The effect of IFN-λ1 to keratinocyte proliferation was measured by Cell Counting kit (CCK-8) assay. Keratinocytes were seeded in a 96-well plate (about 3 × 10³ cells per well). After 24 h incubation, IFN-λ1 at the concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml (Pereprotech, AF-300-02L, United States) were added to the culture media. 10 μl of TransDetect® Cell Counting Kit (CCK) (Transgen, FC101-01, China) was added to each well at the time of 0, 24, 48, and 72 h, respectively. After 1 h incubation, the absorbance at 450 nm was measured by the Multiskan™ FC Microplate Photometer (Thermo Scientific, 51119180, Belgium). Cell colony formation assay was also performed to verify the effect of IFN-λ1 on keratinocyte proliferation. Keratinocytes were planted to 6-well plate at 2,000 cells each well and cultured with various concentration of IFN-λ1 for 15 days. Phosphate buffered saline containing 5% trehalose (5% trehalose-PBS) (MultiSciences, 79-PD0021, China) was used as negative control. After 15 days culture, the cells were fixed with formaldehyde, stained with 0.1% crystal violet, washed with 33% glacial acetic acid and measured with a Multiskan FC microplate photometer (Thermo Scientific, 51119180, Belgium).

Inhibition of Staphylococcus aureus Biofilm Formation
S. aureus samples were incubated with MIC and sub-MIC of the examined agents in Tryptic soy broth with 2% glucose at 37 °C for 24 h. After incubation, the plate was washed twice with sterile PBS and fixed with methanol for 10 min. Methanol was removed by pipetting and the plate was air-dried. The biofilms were stained by using 0.1% crystal violet (Sigma Aldrich, Darmstadt, Germany) for 30 min, water washed and air dried, and the stain was dissolved in 96% ethanol (Zorka, Sabac, Serbia). The absorbance was read at 570 nm on a Multiskan™ FC microplate photometer (Thermo Scientific™, Waltham, MA, USA) and the results are presented as the inhibition of biofilm formation (%) [27 (link)].
Inhibition of the formed Staphylococcus aureus biofilm
The S. aureus inoculum was incubated in a microtiter plate with an adhesive bottom for 24 h at 37 °C. After incubation, the plate was washed 3× with saline and the adhered cells were treated with MBC of extracts for 30 s. After washing, addition of methanol, drying, staining with crystal violet dye and addition of ethanol, the absorbance was read at a wavelength of 570 nm on an automated Elisa reader (Multiskan™ FC microplate photometer, Thermo Scientific), and the percentage of biofilm diminishing was calculated [28 (link)].