Prolonged gold anti fade reagent

The cytocompatibility of DMS was assessed by performing the DAPI staining of seeded cells, and the viability of the seeded myoblast cells on the DMS was assessed by performing the live-dead staining using Calcein AM and propidium iodide (PI). Before cell seeding, mushroom scaffolds were sterilized using 70% ethanol for 30 min, followed by thorough washing with 1xPBS thrice. Monolayer myoblast cells were used as control. Furthermore, 5 × 10⁵ myoblast cells were seeded onto DMS. After 72 h, cells were washed with 1× DPBS and stained with 1 µg/mL DAPI for 10 min. Cells were rinsed with 1× DPBS thrice, mounted with prolonged gold antifade reagent (Invitrogen; cat no. P10144), and captured under a Zeiss confocal microscope.
For viability (live/dead staining), 5 × 10⁵ myoblast cells were seeded onto DMS, and after 72 h, monolayer myoblast and cell-seeded DMS were rinsed with 1× PBS followed by incubation with a staining solution containing 2 µM Calcein AM and 1 µM PI for 30 min. in the dark at room temperature; after 20 min incubation, 1 µg/mL DAPI was added to the staining solution. After incubation, the staining solution was removed, and monolayer myoblast cells and scaffolds were rinsed with 1× PBS and mounted with a prolonged gold antifade reagent (Invitrogen; cat no. P10144). Images were captured under the Zeiss confocal microscope (Jena, Germany).

Following drug treatment, cells were rinsed with PBS and fixed with ice-cold 4% buffered formalin for 10–15 min. Cells were then washed 3X with PBS. Later cells were permeabilized using 0.02% triton 100X and subsequently blocked with 10% goat serum in PBS followed by overnight incubation at 4 °C with primary antibodies in blocking buffer (1:100–150) reactive against human or mouse proteins (Glut1, Nrf2, Nqo1 and Slc40a1). Next day, cells were washed with blocking buffer and incubated for 1 h with Alexa Fluor 488 or 555-conjugated anti-rabbit or anti-mouse antibodies (1:1000) at room temperature. Cells were washed with PBS, dried at room temperature and cover-slipped with DAPI in Prolonged Gold Anti-fade reagent (Invitrogen, Carlsbad, CA). Images were captured using an EVOS XL digital inverted fluorescence microscope at 40 × (Advanced Microscopy Group - AMG, Cat #AME3301).