Replication Data for: lactate of "Replicative Senescence in Mesenchymal Stem Cells: An In Vitro Study on Mitochondrial Dynamics and Metabolic Alterations"

The luminescence-based Lactate-Glo™ Assay (J5021, Promega) was used to quantify intracellular lactate. Specifically, lactate dehydrogenase uses lactate and NAD+ to produce pyruvate and NADH. In the presence of NADH, a pro-luciferin reductase substrate is converted by reductase into luciferin, which is subsequently used in a luciferase reaction to generate light. Therefore, the light emission is proportional to the amount of lactate in each sample. bMSCs were seeded in a 6-well plate (Costar, Sigma-Aldrich) (1.5 × 105 cells/well) and, after 3 days, were detached from the plate and counted for normalization. Cells were then incubated for 5 min at room temperature with Inactivation Solution (0.6 N HCl in H2O) which rapidly stops metabolism, lyses the cells, inhibits activity of endogenous proteins and destroys reduced NAD(P)H dinucleotides. After inactivation, Neutralization Solution (1 M Trizma®) was added to each sample. The samples were aliquoted in duplicate into a 96-well white plate (Costar, Sigma-Aldrich). At the same time, Lactate Detection Reagent was prepared according to the manufacturer’s instructions and added to each well. The plate was incubated for 1 h at room temperature in the dark. Luminescence was measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). The results were normalized to the cell number.