Flexstation 3 multi mode microplate reader

Pectoralis major muscle biopsies from n = 13 female patients with BC and n = 10 control breast surgical patients were snap-frozen and stored at −80 °C until processing as described under “Western blotting analysis.” All available snap-frozen muscle biopsies were included in analysis of ATP content. ATP content was quantified using the ENLITEN^® ATP Assay System Bioluminescence Detection Kit (Promega, Wisconsin, USA) according to manufacturer’s protocol, using the manufacturer’s recommended standard curve for calculation of absolute ATP content from luminescence intensity and The FlexStation^® 3 Multi-Mode Microplate Reader (Molecular Devices^®, CA, USA). ATP content was normalized to protein concentration, quantified using the DC^™ Protein Assay (Bio-Rad, CA, USA).

First, we used the same FS-3 assay as described above, but instead of plasma 10 μL of human recombinant ATX (3, 10 and 30 nM) (Daresbury Proteins Ltd, Warrington, UK) was incubated with FS-3 substrate ± IND (30–300 μM).
In addition, the effect of IND on human recombinant ATX-induced hydrolysis of LPC 18:1 was assessed with the Amplex Red choline release assay. ATX was generated in-house, as described before [43 (link)]. Triplicate wells were loaded with 60 μL of reaction cocktail in ATX assay buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂, 30 μM BSA, pH 7.4), with of 10 μM Amplex Red, 1 U/mL horeseradish peroxidase (both from Thermo Fisher Scientific, Waltham, MA, USA) and 0.1 U/mL choline oxidase (MP Biomedicals, Irvine, CA, USA), resulting in an overall concentration of 100 μM LPC 18:1 (Avanti Polar Lipids, Alabaster, AL, USA) with 10 nM ATX ± IND (30–300 μM). The fluorescence (λ_excitation = 560 nm and λ_emission = 590 nm) was recorded every 2 min by a FlexStation 3 Multi-Mode Microplate Reader for 240 min (Molecular Devices, San Jose, CA, USA).

Diaphragm muscles were excised, carefully cleaned to remove attached tendons, weighed, and snap frozen in liquid nitrogen. Collagen content was measured using a hydroxyproline assay kit (MAK008; Sigma-Aldrich). Briefly, diaphragm muscles were homogenized in water and hydrolyzed overnight in 6 M hydrochloric acid at 110°C. 10 µl of homogenate was then transferred to a 96-well plate and incubated at 60°C to dry the sample. Dried samples were resuspended in chloramine T/oxidation buffer mixture, incubated at room temperature for 5 min, mixed with DMAB-perchloric acid/isopropanol reagent, and then incubated for 90 min at 60°C. Sample absorbance was measured at 560 nm using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). Absorbances (ODs) obtained for each sample were referred against a standard curve to quantify the amount of hydroxyproline, which was then normalized to milligrams of tissue wet-weight.

To investigate the ability of idebenone to modulate LPS-evoked intracellular ROS levels, BV2 microglial cells or mouse primary astrocytes were treated as described above and subsequently incubated with 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen, catalog number: D399, Waltham, MA, USA) for 40 min at 37°C. After washing the cells twice with PBS, the fluorescence intensity (excitation 485 nm, emission 535 nm) was measured and analyzed by using a FlexStation 3 Multi-mode Microplate Reader (Molecular Devices, Jan Jose, CA, USA).

Glucose uptake was measured on slices using the fluorescent glucose analogue 2-NBDG according to [14 (link)]. After the end of the incubation, one slice of each treatment was carefully relocated to a tube containing aCSF supplemented with 2-NBDG (30 µM) and maintained in the media for 15 min. Following 2-NBDG uptake, the slices were washed with aCSF and homogenized in 30 mM Tris-HCl (pH 7.4). Homogenates were centrifuged at 3000× g for 10 min and supernatant fluorescence was measured in FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). One 2-NBDG uptake sample from experiments with K⁺ and pilocarpine is missing because it was lost during processing.

ELISA was performed on culture media from co-cultured U87 + BMM cells to determine the secretion of cytokines using ELISA kits purchased from Thermo Fisher Scientific (Waltham, MA). Cells were prepared and transfected as written in “Flow cytometry” section, and ELISAs were performed according to the manufacturer’s protocol. The data was collected on a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at 450 nm and 570 nm, and the background absorbance taken at 570 nm was subtracted from the absorbance read at 450 nm.