Tyrisin

Primary cultured cardiomyocytes (CMs) were isolated from the heart of 2-day-old Sprague-Dawley rats by a typical method reported by our group^{1 (link)}. In short, the heart was dissected and was separated into a single-cell suspension with 0.1% type II collagenase and 0.25 % Tyrisin (Sigma). The suspension cells were then centrifuged for 5 min at 900 g and then these cells were cultured on plate for 2 h. Finally, the non-attached CMs were collected through centrifugation.
To evaluate the biocompatibility of hydrogels, live-dead staining and CCK-8 test were performed on CMs seeded on the CPA, CPAM and CPAMC hydrogel respectively. For immunofluorescence staining, the samples were incubated in primary antibodies overnight at 4 °C, including mouse anti-α-actinin (1:250), mouse CTNT (1:250) and rabbit anti-CX 43 (1:1000), and then treated with Alexa Fluor-488 Donkey anti-mouse IgG (H&L) (1:500) or Alexa Fluor-568 donkey anti-rabbit IgG (H&L) (1:500) for 1 h. The stained samples were further incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 30 min and observed using confocal microscopy (Zeiss Zen software (ver. 3.2)).

DRG neurons from SD rats (60–80 g) were dissociated as previously described [24 (link)]. Briefly, L4–L6 DRGs were excised in ice-cold DMEM/F12 medium (10565018, GIBCO, USA) and mechanically dissociated. After digested with tyrisin (T9201, Sigma, USA) and collagenase (C9891, Sigma, USA) for 30 min in 37 °C, DRG neurons were seeded onto cover slips coated with poly-l-lysine (P7890, Sigma, USA) in a humidified atmosphere (5% CO₂, 37 °C) for up to 4 h and then were used for patch-clamp recording.