Axio observer z1 microscope system

Transfection of siRNA or DNA constructs into HeLa cells or COS7 cells and immunofluorescence were performed as described (Guo et al., 2013 (link)). Cells growing on coverslips were fixed in 4% PFA for 20 min then washed with PBS and incubated with permeabilization buffer (2.5% FBS, 0.1% TX-100, 0.2 M Glycine in PBS) at RT for 30 min. Then cells were sequentially incubated with primary antibody and secondary antibody diluted in permeabilization buffer for 30 min. Each antibody incubation was following by five times wash with PBS. After staining, cells were fixed again in 4% PFA for 20 min and then washed with PBS. Surface labeling procedure was described (Ma et al., 2018 (link)). Briefly, to label surface-localized Frizzled6 containing an HA tag in its extracellular domain, HeLa cells expressing HA-Frizzled6 were incubated with the mouse anti-HA antibody (1:200) in PBS containing FBS (250 μl in 10 ml) for 40 min on ice. After several washes with PBS, cells were fixed for 15 min with 4% paraformaldehyde in PBS and then a normal immunofluorescence procedure was performed. Fluorescent images were acquired with a Zeiss Axioobserver Z1 microscope system.

For testing cell viability, the device was stained with 300 uL of CalAM (2 mM) and EthD-1 (4 mM) solution (L3224, Thermofisher Scientific) for 1 h to allow for diffusion into the collagen scaffold, and rinsed with PBS twice (10 min each). Then the device was immediately imaged using confocal microscopy (Axio Observer Z1 Microscope System, Zeiss, Oberkochen, Germany). Green fluorescent cells (EGFP positive) were counted as live cells and red cells were counted as dead cells.

30 μl of rhodamine 6 G (Sigma) at a concentration of 10⁻⁴ g/ml^{44 (link)} was added into one channel of each device. Devices were incubated at 37 °C for 0, 1, 4, 8 or 24 hr. The devices were then immediately imaged with a confocal microscope (Axio Observer Z1 Microscope System, Zeiss). The images were then processed linearly between the channels to measure average intensity and thus obtain an intensity profile. Similarly, 30μl of fluorescently labeled EGF (Biotinylated, complexed to Alexa Fluor™ 647 Streptavidin, ThermoFisher Scientific) at a concentration of 10μg/ml was added to one channel and examined at 0, 1, 4, 8, 24 hrs of diffusion (Fig. S4).