Amplite colorimetric l lactate assay kit

Extracellular lactate was measured using an Amplite Colorimetric l-Lactate Assay Kit (AAT Bioquest, #13815). Briefly, supernatants from confluent flasks were diluted at 1:100, and 50 µL of the diluted media was added to 50 µL NAD containing assay buffer in a 96-well plate. The plate was incubated at room temperature for 2 h and absorbance was measured using a CLARIOstar high-performance monochromator multimode microplate reader at 575 nm/605 nm. l-lactate standard curve is used to determine lactate concentrations in the samples.

Total cellular ATP content was measured using an ATPlite assay kit (PerkinElmer) according to manufacturer’s instructions. Briefly, 15 000 cells were counted and used to evaluate ATP content in iPSC, whereas 20 000 neuron progenitor cells were seeded in a 96-well plate and cultured as normally until day 30, when cells were lysed on the plate prior the assay. Six wells per cell line were used. Luminescence was monitored with an EnSpire (PerkinElmer) microplate reader with 0.1 s measurement time and normalized to the cell count (iPSC) or protein levels (MN). Intracellular and extracellular lactate levels were measured using Amplite™ Colorimetric L-Lactate Assay Kit (AAT Bioquest) according to the manufacturer’s protocol. Briefly, 200 000 neuron progenitors were seeded and cultured on 12-well plates and cultured as normally until day 30. Then, 200 μl of the media was used for the extracellular lactate measurement and cells were harvested and lysed for the intracellular lactate measurement. Three wells per cell line were used. Extracellular samples were diluted 1:10 in PBS and all samples were filtered through a 10 kDa MW spin filter (Millipore, Darmstadt, Germany). Fluorescence was recorded at Ex/Em = A540 nm/A590 nm using an EnSpire (PerkinElmer) microplate reader and normalized to protein levels.

Dried PLA or PLA/DLC membranes were cut into disks of 20 mm diameters, and the initial weight was recorded. Sliced membranes were immersed in 25 mL of pH 8.0 Tris buffer containing 10% esterase concentration and 10 mM calcium chloride to imitate the biodegradation environment of tendon injury. Then, the reaction system was sealed and stored at 37 °C. Degraded weighted loss was calculated with dry weight as 0, 1, 4, 6, 10, 14, 21, and 28 days, and a weight-time curve was drawn.
The degraded reaction solution was obtained for measuring lactic acid content at each time point, and lactic acid concentration was measured following the instructions of Amplite™ Colorimetric L-Lactate Assay Kit (Cat.13815, AAT Bioquest, USA). The concentration of released lactic acid was calculated, and a content-time curve was drawn.