Fluostar omega

Isolated colon mitochondria were homogenized in 9 volumes of 100 mM Tris and 4 mM EDTA buffer, pH 7.75, preheated to 95 °C. Samples were incubated at 100 °C for 2 min and centrifuged at 1000×g for 2 min at 4 °C. The supernatant (50 µl) was used to determine ATP with 1 volume of reaction buffer. Luminescence was measured in 96-well plates with FLUOstar Omega (BMG Labtech) using ATP Bioluminescence Assay Kit HS II (Roche, 11699695001) following the manufacturer’s instructions.
Proteins from isolated colon mitochondrial were precipitated in 6 volumes of 6% perchloric acid and neutralized with KOH for the determination of ADP and AMP using ADP Assay Kit (MilliporeSigma, MAK133) or AMP Assay Kit (Abcam, ab273275), respectively, following the manufacturer’s instructions. For determination of total ADP and ATP as well as AMP in the cells, the same ADP Assay Kit (MilliporeSigma, MAK133) or AMP Assay Kit (Abcam, ab273275), respectively, were used. Luminescence and absorbance were measured in 96-well plates with FLUOstar Omega (BMG Labtech).
The adenosine released into the cell culture medium at 24 h lines was determined using the fluorometric Adenosine Assay Kit (Abcam, ab211094) following the manufacturer’s instructions. Fluorescence was measured in 96-well plates with FLUOstar Omega (BMG Labtech).

The culture medium was removed after 3 and 7 days of cell culture and the samples were transferred to new 12-well plates; subsequently, 10% PrestoBlue solution was added (5 mg/mL in DMEM; Thermo Scientific, Oxford, UK) and the multi-well plates were incubated at 37 °C for 2 h. After the supernatant removal, the solution was transferred in 96-well plates (0.2 mL) and quantified with a spectrophotometer working at 560 nm; a Filter-based FLUOstar^® Omega multi-mode reader (FLUOstar^® Omega, BMG Labtech, Ortenberg, Germany) was used. PicoGreen^® dsDNA reagent purchased from Invitrogen (Carlsbad, CA, USA) was employed to calculate the number of the cells for each sample to make a correct normalisation of the fluorescence values. The scaffolds were carefully washed with PBS after each culturing period, incubated for 3 h at 37 °C for 3 h and then frozen at −80 °C overnight in ultra-pure water (1 mL) in order to ensure cell lysis. The assay was carried on according to the protocol of the manufacturer. Fluorescence was measured at an excitation and emission wavelength of 485 and 528 nm, respectively.

Standard curve preparation: hADSCs at passages 4–6 were seeded in 100 µL of culture medium to 96-well plate in following densities: 250, 500, 750, 1000, 2000, 5000, 10,000, 15,000 or 25,000/cm². After 24 h, 10 µL of WST-8 solution (The Cell Counting Kit 8; Dojindo, Kumamoto, Japan) was added to each well and incubated for 2 h at 37 °C. The absorbance was measured at optical density (OD) 450 nm using the FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).
hADSCs at passages 4–6 were seeded in 100 µL of culture medium with compact manganese alginate particles at 10% concentration or without (control) 96-well plate in density 3000 cells/cm². After 24 or 48 h, 10 µL of the WST-8 solution was added to each well and incubated for 2 h at 37 °C. The absorbance was measured at OD 450 nm using the FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).