Multiskan go

RAW 264.7 cells were seeded in 96-well plates at a density of 5 × 10⁵ cells/mL. After 12 h, the cells were treated with 1 µg/mL of LPS and tested samples, followed by additional incubation for 24 h at 37 °C. MTT stock solution (2 mg/mL) was added to wells for a total reaction volume of 100 µL. After 4 h incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in DMSO (100 µL), and the absorbance was measured with the wavelength of 490 nm by a microplate reader (Multiskan GO, Thermo Scientific). The data were expressed as mean percentages of the viable cells compared to the respective control. After pre-incubation of RAW 264.7 cells (1.5 × 10⁵ cells/mL) with 1 µg/mL LPS and samples at 37 °C for 24 h, the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Briefly, cell culture medium (50 µL) was added with Griess reagent (100 µL) and incubated at room temperature for 10 min. The absorbance was measured by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA) at 540 nm wavelength.

The assays for anti-inflammatory activity were performed as described previously [38 (link)]. RAW 264.7 cells were seeded in 96-well plates at a density of 5 × 10⁵ cells/ mL. After 12 h, the cells were treated with 1 µg/mL of LPS and tested samples, followed by additional incubation for 24 h at 37 °C. MTT stock solution (2 mg/mL) was added to wells for a total reaction volume of 100 µL. After 4 h incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in DMSO (100 µL), and the absorbance was measured with the wavelength of 490 nm by a microplate reader (Multiskan GO, Thermo Scientific). The data were expressed as mean percentages of the viable cells compared to the respective control. After pre-incubation of RAW 264.7 cells (1.5 × 10⁵ cells/mL) with 1 µg/mL LPS and samples at 37 °C for 24 h, the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Briefly, cell culture medium (50 µL) was added with Griess reagent (100 µL) and incubated at room temperature for 10 min. The absorbance was measured by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA) at 540 nm wavelength.

To assay complex I activity, wildtype and Cnhsf3∆ strains were cultured separately overnight and grown to an OD₆₀₀ of 0.8, then 100 μl of the resultant culture was mixed with 20 μl CellTiter 96® AQueous One Solution Reagent (Promega), which contains a novel tetrazolium compound, MTS. The mixture was placed in a 96-well assay plate and incubated at 30 or 40 °C for 1 h. NADH levels were determined by measuring absorbance at 490 nm using a MultiskanGO microplate reader (Thermo). All data were normalized using the number of colony-forming units (CFUs).
To monitor the ATP level, cells were prepared as described above. Strains were incubated at the indicated temperature for 30 min, then were disrupted using glass beads and a Bio-Spec bead beater for 3 rounds of 55 s each, resting on ice for 1 min between rounds. ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay (Beyotime) according to manufacturer instructions. The luminescent signal was measured using a Synergy H4 microplate reader (BioTek). All data were normalized to the protein concentration in the sample, which was determined using a MultiskanGO microplate reader (Thermo). Three independent experiments were performed, each including four technical replicates, and a representative data set is presented.

The total phenolic content of CLE samples was determined by the Folin–Ciocalteu assay (ISO 14502-1, 2005), and gallic acid was used as the standard. CLE (500 μL) samples were mixed with 2.5 mL of 10% (w/v) Folin–Ciocalteu reagent and 2 mL of 7.5% (w/v) sodium carbonate. The mixtures were stirred and incubated in darkness for 1 h at room temperature (25 °C). The absorbance of all the CLE samples was measured at 765 nm using a microplate spectrophotometer (Thermo Fisher scientific, Multiskan GO, USA) [31 (link)]. The TPC was expressed as mg gallic acid equivalent (mg GAE/g) of the dry extract [32 (link)].
The free radical scavenging activities of CLE samples were analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [33 ]. A DPPH solution (60 mM) was prepared by dissolving 0.00236 g in 95% ethanol (v/v). The DPPH solution was mixed with CLE samples (50 μL). Trolox (10,000 μM) was used as a standard solution, and methanol was used as a blank. The mixtures were left at room temperature for 30 min. The absorbance was measured at 517 nm using a microplate spectrophotometer (Thermo Fisher scientific, Multiskan GO, USA) [31 (link)]. DPPH activities of CLE samples were expressed as µmol Trolox equivalent (TE)/g of the dry extract [34 (link)].

Cell viability was measured using the conventional MTT assay. RAW 264.7 cells were seeded in 96-well plates at a density of 1.5 × 10⁵ cells/mL. After 12 h, the cells were treated with LPS (1 µg/mL) and samples, followed by additional incubation for 24 h at 37 °C. MTT stock solution (2 mg/mL) was added to wells for a total reaction volume of 110 µL. After 4 h incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in 50 µL of DMSO, and the absorbance was measured using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA) at the wavelength of 490 nm. Relative cell viability was evaluated based on the quantity of MTT converted to the insoluble formazan salt. The optical density of formazan generated in the control cells represented 100% viability. The data were expressed as mean percentages of the viable cells compared to the respective control.
After pre-incubation of RAW 264.7 cells (1.5 × 10⁵ cells/mL) with LPS (1 µg/mL) and samples at 37 °C for 24 h, the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production. Briefly, 50 µL of cell culture medium were mixed with 100 µL Griess reagent, and incubated at room temperature for 10 min. The absorbance was determined at 540 nm wavelength with a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Thiobarbituric acid-reactive substances (TBARS) were used to determine lipid peroxidation. Briefly, samples were first centrifuged at 12,000× g at 4 °C for 10 min, then 250 μL of the sample, 10 μL of 4.5 mM butylated Hydroxytoluene (BHT), and 200 μL of 30% trichloroacetic acid (TCA) were added to 1.5 mL Eppendorf tubes. These were subsequently placed in a boiling water bath (100 °C) for 15 min, and centrifuged at 15,000× g at room temperature for 2 min. Next, 400 μL of supernatant and 400 μL of 0.23% thiobarbituric acid (TBA) were pipetted into the cryotubes and boiled in a 100 °C water bath for 30 min. The samples were cooled down for 5 min and pipetted in duplicates of 200 μL into a 96-well plate. TBARS was then determined in a microplate reader at 540 nm (Multiskan Go, Thermo Scientific, Waltham, MA, USA) [29 (link)]. Carbonyl assay was used to determine oxidative damage to proteins and the absorbance was read at 360 nm (Multiskan Go, Thermo Scientific, Waltham, MA, USA) [30 (link)].