Spectramax i3x system

Aliquots of 3 × 10³ MSCs or MEFs were cultured in 96-well plates for the following treatment. After incubation periods of 1 d, 2 d, 3 d and 4 d, Cell Counting Kit-8 (Dongjindo Molecular Technologies, Inc., Tokyo, Japan) was used for calculating cell viability. Absorbance was measured at 450 nm using a Molecular Devices SpectraMax i3X system. For the next treatment, 1 × 10⁵ MEFs were cultured in 6-well plates. After 24 h of culture, the MEFs were harvested and subjected to Ki67-FITC staining. Flow cytometry was performed to examine the expression of Ki67 in MEFs using Accuri C6-BD. The results were analyzed using Flow J 10.4.

After experimental myotube incubation, cells were lysed on ice with a homogenization buffer (50 mmol Tris-HCl/L, pH = 7.5, 1 mmol EDTA/L, 5 mmol MgCl₂/L, 0.1 mmol dithiothreitol/L, 10% glycerol). After centrifugation and before measurement of protease activities, protein content was measured by spectrophotometry (Nanophotometer NP 80, Implen^®, Germany). Chymotrypsin-like activity of the proteasome fraction was measured using the fluorogenic substrate SUC-LLVY-AMC [succinyl-Leu-Val-Tyr-7-amido-4-methylcoumarin; AMC)] (Sigma-Aldrich^®) [37 (link)]. Then, 10 µL of the supernatant fluid (∼10 μg protein) was incubated in 100 μL of buffer (50 mmol Tris-HCl/L, pH 7.5, 1 mmol ATP/L, 5 mmol MgCl₂/L, and 150 μmol LLVY/L) in microplates. Standard curves were prepared using the AMC. Fluorescence was measured continuously over 1 h at 37 °C on a SpectraMax^® i3X system (Molecular devices^®) at λex = 380 nm and λem = 460 nm. Proteolytic activity was calculated from the increment of the curves from samples and standards and were expressed as pmol of AMC released/μg protein per minute.