Spectrostar nano microplate reader

RAW 264.7 cells were seeded at a density of 2 × 10⁵ cells/well in a 96-well plate and cultured for 2 h. For optimization, the cells were treated with LPS (10 ng and 100 ng) and IFN-γ (5 and 10 U/mL) separately. Various combinations were used to determine the noncytotoxic concentration necessary for stimulating the RAW 264.7 cells. In another experiment, the cells were treated with increasing concentrations of DOX (0.005, 0.01, 0.05, 0.1, or 0.5 µM). Additionally, increasing concentrations of herbal treatments CUR, RES, and SFN (5, 10, and 20 µM) were tested in the presence or absence of LPS/IFN-γ. Based on the optimization results, RAW 264.7 cells in all subsequent experiments were stimulated with 10 ng/ml LPS plus 10 U/mL IFN-γ plus 0.1 µM DOX and coincubated with CUR, RES, or SFN at concentrations of 5, 10, or 20 μM for 24 h at 37 °C in a humidified incubator with 5% CO₂. Cell viability was analyzed by MTT colorimetric assay as previously described¹⁵ . After 24 h of incubation, the medium was discarded and replaced with 1 mg/ml MTT dissolved in serum-free DMEM. After 2 h of incubation, the formazan crystals that had formed were dissolved in isopropanol. Then, the absorbance was measured at 540 nm using a Nano SPECTROstar microplate reader (BMG LABTECH, Ortenberg, Germany), and the percentage of viable macrophages relative to the control was calculated.

The colorimetric MTT assay was done to determine the nontoxic concentration of propolis as previously described [25 (link)]. RAW 264.7 macrophages were exposed for 24 h to propolis at increasing concentrations of (3.125 to 400 μM) alone or with LPS/ IFN-γ (10 ng/mL/10 U/mL). After 24 h, a new serum-free medium with 1 mg/mL MTT was added. The formed formazan crystals were dissolved with isopropanol and the OD was measured at 540 nm using Nano SPECTROstar microplate reader (BMG LABTECH, Ortenberg, Germany). Cell viability was calculated as the percentage of viable macrophages relative to the control.

Total protein content in the cytoplasmic and nuclear fractions was quantified using the Pierce™ BCA Protein Assay Kit. In a 96-well microtiter plate, 25 μL of each sample and standard dilution were added to the designated wells. Every sample or standard dilution was assayed in triplicates. In each well, 200 μL working BCA reagent were added, the plate was thoroughly mixed on an orbital shaker for 30 s and then incubated at 37 °C for 30 min. The plate was then cooled down, and the absorbance of the formed colored complexes was measured at 562 nm using NanoSPECTROstar microplate reader (BMG LABTECH, Ortenberg, Germany). Sample concentrations were computed from the line equation generated by plotting OD values for varying concentrations of a bovine serum albumin standard.

Pre-coated micro-ELISA plates from Elabscience® were used to quantify the protein expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in the supernatant of SIM-A9 cells stimulated with LPS in the presence of GSK3 inhibitors or SFN, following the treatment protocol above. In short, the cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/mL. Following overnight incubation, the cells were pretreated with GSK3 inhibitors (20 μM) or SFN (5 μM) for 2 h. The media were then removed and replaced with fresh media containing 100 ng/mL of LPS while maintaining the concentration of GSK3 inhibitors at 20 μM and SFN at 5 μM. The culture media were collected 24 h later, centrifuged at 1000 × g for 20 min at 4 °C and the supernatant was transferred to clean microcentrifuge tubes. For IL-6 and TNF-α, the supernatant was diluted in a ratio of 1:20 in buffered sample diluent, whereas undiluted samples were used for the detection of IL-1β. Optical density was measured at 450 nm using a NanoSPECTROstar microplate reader (BMG LABTECH, Ortenberg, Germany).

The lactate dehydrogenase CyQUANT LDH cytotoxicity assay kit (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer’s instructions. Briefly, the lactate dehydrogenase (LDH) released into the medium was quantified. The culture medium was set aside to measure extracellular LDH activity. To estimate the intracellular LDH activity, the cells were lysed using the assay buffer provided in the kit. The assay buffer facilitated the release of intracellular LDH, allowing for its activity to be measured. The SPECTROStar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany) was used to detect the OD values at 490 and 680 nm.
% Cytotoxicity was calculated using the following formula: $$\% \mathrm{Cytotoxicity}=\frac{\left(\mathrm{Compound} \mathrm{treated} \mathrm{LDH} \mathrm{activity}-\mathrm{DMEM} \mathrm{media} \mathrm{LDH} \mathrm{activity}\right)}{\left(\mathrm{Lysed} \mathrm{cells} \mathrm{LDH} \mathrm{activity}-\mathrm{DMEM} \mathrm{media} \mathrm{LDH} \mathrm{activity}\right)}$$
At least 3 replicates were conducted in each experiment.

The thiol group content was measured spectrophotometrically (the SPECTROstar Nano Microplate Reader – BMG LABTECH Germany) by absorbance at 412 nm with Ellman’s reagent: 5,5′-dithio-bis-(2-nitrobenzoic acid). The thiol group concentration was calculated using a molar extinction coefficient (ε = 13,600 M⁻¹cm⁻¹) (Ando and Steiner, 1973a (link),b (link); Bartosz, 2008 ). The level of thiol groups was expressed as nmol thiol groups/mg of plasma protein.

Samples of plasma or erythrocytes were transferred to an equal volume of cold 20% (v/v) trichloroacetic acid in 0.6 M HCl and centrifuged at 1200 × g for 15 min. One volume of clear supernatant was mixed with 0.2 volume of 0.12 M thiobarbituric acid in 0.26 M Tris (pH 7.0) immersed in a boiling water bath for 15 min. and then absorbance was measured at 535 nm (the SPECTROstar Nano Microplate Reader – BMG LABTECH Germany) (Wachowicz, 1984 (link); Bartosz, 2008 ). The TBARS concentration was calculated using the molar extinction coefficient (ε = 156,000 M⁻¹cm⁻¹).