Clariostar plate reader

Cells were seeded in 96-well plates, as 2 × 10³ cells/well and treated with 3 or 10mM βHb in KD media. BrdU proliferation assay (Cat#QIA58. Sigma-Aldrich) was performed after 96 h of incubation. BrdU 1000x was added to a concentration of 1x. The plate was incubated for 4 h. The level of BrdU was measured by absorbance at 450-540 nm using the ClarioStar plate reader (BMG LABTECH).
For starvation conditions the cells were plated at 70% confluence into 96-well plates. After overnight attachment, the media was changed to starvation media (DMEM no glucose, no L-glutamine, no phenol red, no sodium pyruvate) and the cells were treated with βHb 10mM alone or in combination with one of the nutrient components (0.25mM pyruvate, 1mM glucose and 10mM L-glutamine). On the 6th day, the BrdU label was added to each well and incubated for 24 h. The levels of antibody bound to the DNA-incorporated BrdU was detected by analysing the media color change caused by an enzymatic reaction and measured as an absorbance at 450-540 nm using the ClarioStar plate reader (BMG LABTECH).

HT1080-ISRE-mCherry cells were plated in 6-well plates at 2 × 10⁴ cells/well. The next day, DMEM was replaced with DMEM containing ALOS4 or saline as control and cells continued to grow for 48 h. Following the 48 h incubation, 25 µg/well of Poly I:C was added for an additional 16 h incubation. Following treatment, cells were subjected to fluorescence quantification as follows: cells were washed with 1× PBS, trypsinized and resuspended in 300 µL of 1× PBS, and 200 µL of cell suspension was further seeded into 96-well black-walled, clear-bottom plates. The intensity of the mCherry fluorescence signal was detected with multiple reads per well at 574_Ex/610_Em using ClarioStar plate reader (BMG Labtech, Ortenberg, Germany).
HCT116 cells were plated in 96-well plates at 3 × 10³ cells/well. ALOS4, Poly I:C or saline as control were administered with/without PEI—transfection reagent. In order to detect reporter signal, the media were removed, and 30 µL of Bright-Glo™ Luciferase Assay System (Cat# E2610; Promega, Madison, WI, USA) was added. The signal was quantified by ClarioStar plate reader (BMG Labtech) on luminescence parameters.

ATP assays were performed as previously described (Sen et al., 2016 (link)). In short, about 20 thoraces from pupae or adult flies were homogenized in 75 µl of ATP extraction buffer (100 mM Tris-Cl, pH 8.0, 4 mM EDTA, pH 8.0, 6 M guanidine hydrochloride) and used for both ATP and bicinchoninic acid (BCA) assay. ATP concentrations were determined using ATP determination Kit (Invitrogen, Thermo Fischer Scientific, Waltham, MA, United States) as per the manufacturer’s protocol. The luciferase activity was measured in 96-well format using a CLARIOstar plate reader (BMG Labtech Inc, Cary, NC, United States). Protein concentrations were determined using a BCA protein assay kit. 5 µl of diluted samples (1:25) were mixed with 100 µl of BCA reagent (Pierce BCA protein assay Kit, Thermo Fischer Scientific, Waltham, MA) and protein concentrations were measured on a CLARIOstar plate reader (BMG Labtech Inc, Cary, NC, United States). Results were averaged over three technical replicates from each samples and represented as percent ATP concentrations normalized to the protein concentrations.

The levels of the antioxidant PON1 arylesterase activity in serum samples were determined in a similar way to a previously described method [28 (link)]. Serum was diluted 1:80 in salt buffer (20 mM Tris/HCl, 1.0 mM CaCl₂, pH 8.0) and a triplicate of 20 μl diluted serum were added to the wells in a UV-transparent 96-well plate (Sigma–Aldrich,). A total of 200 μl of phenyl acetate solution, containing 3.26 mM phenyl acetate in salt buffer, were added to each well and the absorbance of produced phenol was measured at 270 nm in a Clariostar plate reader (BMG Labtechnologies, Offenburg, Germany). The initial period when the reaction was linear were used for calculation of activity, expressed as U/ml, using an extinction coefficient of phenol of 1310 M⁻¹.cm.

Serum Paraoxonase/Arylesterase 1 (PON1) activity was measured in plasma as previously described [28 (link)]. Briefly, lithium heparin plasma was diluted 1:80 with a salt buffer (20 mM Tris–HCl and 1.0 mM CaCl₂) and a triplicate of 20 μl diluted plasma were added to the wells in a UV-transparent 96-well plate (Sigma–Aldrich). The volume 200 μl of phenyl acetate solution, containing 3.26 mM phenyl acetate in salt buffer, was added to each well and the absorbance of produced phenol was measured at 270 nm in a Clariostar plate reader (BMG Labtechnologies, Offenburg, Germany). The initial period when the reaction was linear was used for calculation of activity, expressed as U/ml, using an extinction coefficient of phenol of 1310 M⁻¹cm⁻¹.