Pico green dye

Pico‐green fluorescent dye (Quant‐iT™ PicoGreen™ dsDNA Assay Kits, Fisher Scientific, USA) was used to determine the cfDNA binding efficiency of the nanosheets. Briefly, calf thymus DNA (1 µg mL⁻¹, Sigma–Aldrich, USA) was mixed with Pico‐green dye (2 µg mL⁻¹) and incubated in TE buffer at 37 °C for 30 min. Then, PGA, M‐PGA‐S, M‐PGA‐M, M‐PGA‐L, PGA‐C12 and PAMAM‐G3 with concentrations ranging from 0.0625 to 32 µg mL⁻¹ were added, and the mixture was incubated at 37 °C for 30 min. The fluorescence intensities were analyzed with 480‐nm excitation and 520‐nm emission wavelengths.

Cell attachment was
assessed at day 1 and proliferation after days
7 and 11 by measuring the DNA content using Picogreen dye (Thermo
Fisher Scientific, USA) as described earlier.^{43 (link)} Briefly, 0.3 mL of lysate solution was prepared using 0.2 mg mL^–1 proteinase-k (Sigma-Aldrich, USA) and 0.02% sodium
dodecyl sulfate (SDS), added to the scaffolds, and incubated at 37
°C for 12 h. Further, lysate solution was collected, freeze–thawed
at −80 °C, and transferred to a 96-well TCP, and an equal
amount of Picogreen dye was added to each well and incubated for 5
min. Fluorescence measurements were recorded at 485 excitations and
520 emission wavelengths in a microplate reader. The dsDNA amount
was calculated using a standard curve plotted by a serial dilution
of a known DNA concentration.
For SEM, cells were fixed on the
scaffolds using a 4% formaldehyde solution (VWR, Sweden) for 30 min,
and scaffolds were then critically dried using a series of alcohols:
30, 50, 70, 90, and 100%. Confocal microscopy (Zeiss LSM 800) was
used to visualize cells in 3D scaffolds. Cells were fixed using 4%
formaldehyde, washed with PBS, and stained with fluorescent dyes.
Nuclei were stained with SYTOX Green (Thermo Fisher Scientific, USA)
in a 1:30000 dilution of stock solution and incubated for 15 min at
RT. The cell cytoskeleton was stained using Alexa Fluor 546 dye (6.6
μM; 30 min; Thermo Fisher Scientific, USA).