Nitroblue tetrazolium nbt

Curcumin extract (90%) was purchased from Sisco Research Laboratories (SRL) Pvt. Ltd., (Mumbai, Maharashtra, India). High Capacity cDNA kit, phosphate buffer saline (PBS), formaldehyde, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), trichloroacetic acid, thiobarbituric acid (TBA), butanol, nitroblue tetrazolium (NBT), disodium hydrogen phosphate, sodium citrate, and hydrogen peroxide (H₂O₂) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse brain-derived neurotrophic factor (BDNF) ELISA kit and mouse CREB ELISA kit were purchased from Ray Biotech (Georgia, GA, USA). Mouse Synapsin ELISA kit and mouse DCX ELISA kit were purchased from Gentaur (Kampenhout, Belgium). RNA Isolation Reagents and TRIzol were purchased from Sigma Aldrich (St. Louis, MO, USA). SYBR Green was purchased from Applied Biosystems, MA, USA. NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), Polytron tissue homogenizer (Thomas Scientific, Swedesboro, NJ, USA), ELISA reader (Trans Asia Pvt. Ltd., Mumbai, Maharashtra, India), and UV spectrometer (Perkin Elmer, Waltham, MA, USA) were used during the conduction of experimental procedures.

ASC-exosomes and adipose serum-deprived stem cells were lysed in 1XPBS containing protease inhibitor cocktail tablets 1X (Roche) for four times at 25 Hz for 10 s. Samples (60 μg per lane) were loaded in a native buffer and separated on a 15% (v/v) non-denaturing polyacrylamide gel for 3 h at 40 mA at 4 °C. The gel was stained for 45 min in 50 mM potassium phosphate (KH₂PO₄) pH 7.4 containing 275 μg/mL nitro blue tetrazolium (NBT) (ThermoFisher), 65 μg/mL riboflavin (Sigma-Aldrich) and 3.2 μL/mL tetra methyl ethylene diamine (TEMED) (Sigma-Aldrich) at room temperature in the dark. Gel was then illuminated for 15 min until sufficient contrast between achromatic zones (dismutase activity) and blue background was achieved and then scanned for documentation. ASC cells extract was used as positive control. An identical gel was stained with Coomassie brilliant blue to verify the equal amount of protein extract loaded among the different samples.