To analyze
cellular cytoskeletal staining and to visualize overall cell morphology
on fibrinogen scaffolds with different topographies, cells were fixated
using 4% (v/v) solution of paraformaldehyde (PFA) in PBS (Biotrend,
Cologne, Germany) for 30 min at room temperature and were analyzed
via fluorescence microscopy as well as SEM imaging.
For cytoskeletal
staining, actin filaments of cells grown on fibrinogen scaffolds for
4 days were stained with phalloidin (ActinRed ReadyProbes Reagent,
Life Technologies Europe BV, Netherlands) and nuclei were stained
with Hoechst H33342 (NucBlue Live ReadyProbes Reagent, Life Technologies)
for 30 min at room temperature using 2 drops/500 μL of PBS.
After washing two times with PBS, stained samples were mounted onto
glass slides with Prolong Gold antifade mounting medium (ThermoFisher)
and cured overnight at room temperature. The specimens were imaged
at 40× magnification in an inverted fluorescence microscope (Ti-E
– V5.30, Nikon, Tokyo, Japan) and appropriate filter settings
(λex = 540 nm and λem = 565 nm for
Actin Red and λex = 330–380 nm and λem = 435–485 nm for H33342).
Fluorescence images
of phalloidin and H33342 stained cells were
analyzed using the open-source software ImageJ provided by the NIH,51 (link) from three independent experiments performed
in triplicate for each substrate type, amounting to nine images analyzed
per sample. Analysis of the cell orientation was performed using the
red and blue channel via the ImageJ plugin OrientationJ and the Origin
2021 software as described earlier.45 (link),47 (link)For
SEM analysis, cells grown on fibrinogen scaffolds for 10 days
were dried with ethanol exchange by gradually increasing the concentration
of pure ethanol on the samples. Samples were subsequently sputter-coated
with gold for 25 s using a sputter coater 108 auto system (Tescan
GmbH, Dortmund, Germany) before SEM imaging with a Zeiss Supra 40
device (Carl Zeiss, Oberkochen, Germany) at an acceleration voltage
of 3 kV.
cellular cytoskeletal staining and to visualize overall cell morphology
on fibrinogen scaffolds with different topographies, cells were fixated
using 4% (v/v) solution of paraformaldehyde (PFA) in PBS (Biotrend,
Cologne, Germany) for 30 min at room temperature and were analyzed
via fluorescence microscopy as well as SEM imaging.
For cytoskeletal
staining, actin filaments of cells grown on fibrinogen scaffolds for
4 days were stained with phalloidin (ActinRed ReadyProbes Reagent,
Life Technologies Europe BV, Netherlands) and nuclei were stained
with Hoechst H33342 (NucBlue Live ReadyProbes Reagent, Life Technologies)
for 30 min at room temperature using 2 drops/500 μL of PBS.
After washing two times with PBS, stained samples were mounted onto
glass slides with Prolong Gold antifade mounting medium (ThermoFisher)
and cured overnight at room temperature. The specimens were imaged
at 40× magnification in an inverted fluorescence microscope (Ti-E
– V5.30, Nikon, Tokyo, Japan) and appropriate filter settings
(λex = 540 nm and λem = 565 nm for
Actin Red and λex = 330–380 nm and λem = 435–485 nm for H33342).
Fluorescence images
of phalloidin and H33342 stained cells were
analyzed using the open-source software ImageJ provided by the NIH,51 (link) from three independent experiments performed
in triplicate for each substrate type, amounting to nine images analyzed
per sample. Analysis of the cell orientation was performed using the
red and blue channel via the ImageJ plugin OrientationJ and the Origin
2021 software as described earlier.45 (link),47 (link)For
SEM analysis, cells grown on fibrinogen scaffolds for 10 days
were dried with ethanol exchange by gradually increasing the concentration
of pure ethanol on the samples. Samples were subsequently sputter-coated
with gold for 25 s using a sputter coater 108 auto system (Tescan
GmbH, Dortmund, Germany) before SEM imaging with a Zeiss Supra 40
device (Carl Zeiss, Oberkochen, Germany) at an acceleration voltage
of 3 kV.
