Opa reagent

Cells were seeded in 10-cm dishes (2−8 × 10⁴ cells/dish) and incubated for 24 h, after which inhibitors were added and cells were allowed to grow for the indicated periods of time. Cells were lysed in lysis buffer (25 mM HEPES, pH 7.6, 150 mM NaCl, 1% NP-40, 2% SDS, and 10% sucrose) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (5 mM NaF, 200 μM sodium orthovanadate, 1 mM sodium molybdate, 2 mM sodium pyrophosphate, and 2 mM disodium beta-glycerophosphate). Protein concentrations were determined using OPA reagent (Thermo), according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE using gels prepared with WIDE RANGE Gel Preparation Buffer (Nacalai tesque, Kyoto, Japan). After electroblotting and blocking with skim milk, membranes were first reacted with a primary and then with appropriate secondary antibodies conjugated to fluorescent dyes. An Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used for signal detection. The primary and secondary antibodies used are listed in Supplementary Table S1. ImageJ v1.49 (http://imagej.nih.gov/ij/) was used for image analysis.

The o-phtalaldheyde (OPA) based assay is considered a conventional technique to quantify the DoFs of photo-crosslinkable biopolymers (Pien et al., 2022 (link)) and has been used to characterize gelatin modification (Yue et al., 2017 (link); Krishnamoorthy et al., 2019 (link)).
OPA reagent (Thermo Fischer, 26025) was warmed at RT before usage. Briefly, different solutions of unmodified gelatin in DPBS (0.02, 0.1, 0.5, 0.75 and 1 mg/mL) were prepared to derive the standard curve. GelMA solutions in DPBS were prepared at 1 mg/mL concentration. After proper dissolution by warming them at 50°C followed by vortexing, they were cooled down to RT and then reacted with OPA on a ratio of (1:2 v/v) for 60 s. A microplate reader (Synergy™ HTX Fluorescence Multi-Mode Microplate Reader) used an excitation/emission of 360/460 nm to measure the fluorescent intensity of the samples after 5 min from the reaction. The DoF was, then, calculated as described in the follow equation 1: $DoF=\left(1-\frac{C_{eq}}{C_{sample}}\right)\bullet 100\%$ Where $C_{eq}$ is the equivalent modified gelatin concentration of the sample determined by the standard curve and $C_{sample}$ is the tested sample concentration.