br Porosity analysis br Sca
2.5. Porosity analysis
Scaﬀold porosity was measured using a modified liquid displace-ment method where isopropanol was used as the displacement liquid . Rectangular samples (10 mm × 10 mm × 5 mm, n = 5) for each scaﬀold composition were prepared, the sample dimensions were measured using a digital micrometer (Mitutoyo), and the scaﬀold vo-lume (Vi) was calculated. The dry scaﬀold weight (Wi) was measured using an electronic analytical balance (ML54T, Mettler Toledo, Swit-zerland). The scaﬀold was immersed in 30 mL of isopropanol with known density (ρi = 0.785 g/mL) under vacuum for 30 min to remove air from the pores. The isopropanol saturated scaﬀold was weighed (Wf) and the porosity was calculated (Eqn. (1)).
75 cell culture flasks until 70% confluency at 37 °C and 5% CO2 in a fully humidified incubator. PC-3 Sorafenib were cultured in F-12K media, while C4-2B and 22Rv1 cells were cultured in RPMI 1640 media, with both media containing 10% FBS and 1% PS. The cells were detached from the culture flasks with trypsin-EDTA, centrifuged at 200 g, and resuspended in fully supplemented media at 1 × 106 cells/mL. The CA scaﬀolds were placed in 12 well tissue culture plates and seeded dropwise with 100,000 cells in 100 μL of fully supplemented media and incubated at 37 °C and 5% CO2 for 1.5 h, then 1.5 mL of fully supple-mented media was added to each well. The scaﬀolds were transferred to new well plates after 24 h to avoid any eﬀects from cells attached to the bottom of the well. Samples were cultured for 15 d and the media was changed every other day. Cell seeding eﬃciency was calculated by detaching the cells remaining in the initial well plates with trypsin-EDTA following the procedure described above and cell number was counted with hemocytometer with n = 4 samples per scaﬀold compo-sition and cell line.
The cell number for the scaﬀold cultures was quantified at 5, 10, and 15 d with the Alamar blue assay following the manufacturer's protocol. Alamar blue solution (10% Alamar blue reagent in fully supplemented media) was added to each well and the samples were incubated at 37 °C and 5% CO2 for 1.5 h. The Alamar blue solution was then transferred to a black 96-well plate to obtain fluorescence values using Cytation 5 cell imaging multi-mode plate reader (BioTek, Winooski, VT) at excitation wavelength of 560 nm and fluorescence emission read at 595 nm. The cell number was calculated using a standard curve prepared with known cell numbers for each cell line.
2.9. Immunofluorescence analysis
Samples were fixed with 3.7% formaldehyde for 1 h at 37 °C, wa-shed with PBS three times and stored in PBS at 4 °C for histology ana-lysis. The samples were embedded in paraﬃn and 5 μm sections were cut and mounted on slides, deparaﬃnized with xylene and ethanol, then rehydrated in TE buﬀer. Antibody solutions were prepared in 10% bovine serum albumin solution in D-PBS.
Primary antibodies used include rabbit monoclonal E-cadherin (1:200 dilution, Cell Signaling Technology, Danvers, MA), rabbit monoclonal pan-actin (1:200 dilution, Cell Signaling Technology), mouse monoclonal cytokeratin 8 (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal androgen receptor (1:50 dilution, Cell Signaling Technology), rabbit polyclonal anti-os-teocalcin (10 μg/mL dilution, Abcam, Cambridge, MA), and rabbit monoclonal phospho-EGF receptor (1:500 dilution, Cell Signaling Technology). The samples were incubated with the primary antibodies overnight at room temperature in a sealed box to avoid evaporation. The samples were washed three times with D-PBS, then the secondary antibodies were added. The secondary antibodies included fluorescein goat anti-rabbit, DyLight 488 horse anti-mouse, biotinylated horse anti-rabbit, and Texas Red streptavidin (all 1:200 dilution, all from Vector Laboratories, Burlingame, CA). The secondary antibodies were in-cubated at room temperature for 20 min then washed with D-PBS, with the biotin and streptavidin antibodies being applied in two incubations. The samples were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). The samples were imaged with a Leica TCS SP5 confocal microscope (Leica, Buﬀalo Grove, IL) using the 405 nm, 488 nm and DPSS 561 nm lasers. Protein expression was quantitated by applying a threshold for each marker based on sec-ondary only controls and average intensity was divided by cell number with Fiji  for at least 6 images per experimental group.