Cytation three microplatereader

Heparin neutralization with compounds
was evaluated using a commercial two-stage kit, Biophen Anti-Xa (221005).
Protein copolymers of different concentrations were first lyophilized
and redissolved in plasma. Dissolved compounds were then added to
the heparin solution in 150 mM NaCl (0.1 mg mL^–1), giving a final heparin concentration of 0.045 IU mL^–1 (12.5 nM) and protein copolymer/heparin mass ratio from 0 to 150
for all protein copolymers. Kit reagents were utilized according to
the manufacturer’s instructions. To run the calorimetric assay,
40 μL of the protein copolymer–heparin solution was added
to a 96-well microplate followed by the addition of 40 μL of
antithrombin and incubation for 2 min. Then, 40 μL of factor
Xa was added and incubated for another 2 min. Afterward, 40 μL
of the factor-Xa-specific chromogenic substrate was added to the solution
and left to react for 2 min. Finally, the reaction was quenched by
introducing 80 μL of 2% citric acid. The absorbance at 405 nm
was recorded immediately using a BioTek Cytation three-microplate
reader. The anticoagulant activity is inversely proportional to the
measured absorption intensity, and the percentage of neutralization
was determined using a calibration curve constructed according to
the manufacturer’s instructions (Figure S4). Measurements were performed using triplicate samples.

To track the migration of patterned REF 2c cells, brightfield live-cell imaging was performed at 37°C, 5% CO₂ using an automated digital microscope with a 10× objective with a gas controller (Cytation three microplate reader, BioTek Instruments Inc, Winooski, VT, USA). Images were collected every 10 min for 24 hr. Acquired brightfield images were merged and corrected for frame drift. To analyze cell migration, individual cell positions were manually tracked using CellTracker software (Piccinini et al., 2016 (link)) implemented in MATLAB (MATLAB R2020a, MathWorks). The average speed for each cell was calculated as the total migration length of each cell divided by the total time. Mann–Whitney test was used to compare the migration length and average speed of the cells since the data were not normally distributed (Shapiro-Wilk test). Statistical differences were defined as where *, p < 0.05; **, p <0.01; ***, p <0.001.