Glycogen colorimetric assay kit

Liver and skeletal muscles were used to investigate whether glycogen contents of these two tissues could enhance by RES-SNEDDS administration. Glycogen levels were measured using a glycogen colorimetric assay kit (BioVision, Milpitas, CA, USA). For each rat, 10 mg of liver and muscle was cut and weighed and then the tissues were rapidly homogenized with 100 µL ice cold glycogen hydrolysis buffer. After centrifugation at 12,000 rpm for 5 min at 4 °C, the supernatant was collected and added into a 96-well plate and brought the volume of 50 µL with glycogen hydrolysis buffer, following by adding 2 µL hydrolysis enzyme mix and 48 µL reaction mix containing glycogen development buffer, development enzyme mix and probe. After incubated at room temperature for 30 min, absorbance was read at 450 nm using a Varioskan Flash plate reader (Thermo Scientific, Waltham, MA, USA).

REH cells (2 × 10⁶) from each cell population were used to measure the glycogen content using a glycogen colorimetric assay kit according to the manufacturer’s instructions (Biovision, Milpitas, CA, USA).

The glycogen levels in soleus muscle samples were measured using a glycogen colorimetric assay kit (BioVision) according to the manufacturer’s instructions. Soleus was selected as the target muscle for this study as studies have shown that soleus muscle is one the key muscle types that can be improved with swimming training in rat model (Forte et al., 2020 (link); Moustafa and Arisha, 2020 (link)).

Glycogen content in the heart tissue was determined using Glycogen Colorimetric Assay kit (BioVision, Milpitas, CA) according to the manufacturer’s protocol. Langendorff-perfused heart tissues (10 mg) were homogenized with 200µl dH₂O (on ice), boiled (100°C, 10 minutes), and centrifuged (18000g, 10 minutes). The supernatants (50 μl) were transferred to a 96-well plate. To hydrolyze the glycogen to glucose, 2μl of Hydrolysis Enzyme Mix were added and incubated (room temperature, 30 minutes). The samples were incubated with 50μl of Reaction Mix (Development buffer 46 µl, Development Enzyme Mix 2μl, OxiRed Probe 2μl) (room temperature, 30minutes), and then, absorbance was measured (optical density at 570 nm).

Glycogen levels were measured in hASCs using the Glycogen Colorimetric Assay Kit (BioVision Inc.,). In total, 3 × 10⁵ hASCs were homogenized with 200 μl of water on ice, and the assay was performed following manufacturer's instructions. A glucose background control was determined and then subtracted from the glycogen readings.

Liver lysate was first normalized for protein concentration using Pierce™ BCA protein assay kit (Thermo Fisher Scientific Inc., Illinois, USA). BUN and LDH activities were measured by a urea nitrogen colorimetric detection kit (Arbor Assays, BioVision Inc., Illinois, USA) and LDH colorimetric assay kit (BioVision Inc., CA, USA), respectively, at 450 nm following the manufacturer's instruction. Glycogen concentration was measured using a glycogen colorimetric assay kit (BioVision Inc., CA, USA) at 570 nm according to manufacturer's manuals.