Librate. The tumor cell suspension medium in the channel was removed 1 hour later and all channels in the device were filled with endothelial cell culture medium. Control experiment with MCF-10A was done following the exact tumor cell seeding protocol. All cultures were kept in a humidified incubator, which was maintained at 37uC and 5 CO2.using OPENLAB 4.0.4 software. Images were later analyzed using MATLAB to calculate fluorescence intensity across the monolayer. To determine the 1655472 diffusional permeability, 25033180 we calculated the distribution of fluorescence intensity change as a function of distance perpendicular to the plane of the endothelial layer. A detailed procedure for measuring permeability has been described previously [24,35,36,37]. Briefly, we used the equation P = D [dC/ dx]/DCec where P is the Chebulagic acid web diffusive permeability (cm/s), dC/dx is the gradient of the dextran concentration, DCec is the concentration difference across the monolayer, and D is diffusion coefficient of dextran.Immunofluorescent Staining and Image AcquisitionAll cells in the device were washed with Phosphate Buffered Saline (PBS) and later fixed with 4 paraformaldehyde for 15 min. After washing twice with PBS, cells were permeabilized with 0.1 Triton-X 100 solution for 5 min and blocked with 5 BSA solution for 5 h. VE-cadherin was labeled with rabbit polyclonal antibody (polyclonal; Alexis Biochemical) at 1:100 dilution and subsequently applied fluorescently-labeled secondary antibody. Cell nuclei were stained with DAPI (Invitrogen) at 1:1000 dilution. All images were obtained using a confocal microscope (Leica) and processed with IMARIS software.Metrics for ExtravasationQuantitative cell counting was performed after immunofluorescent staining. Confocal data were analyzed using IMARIS and its tracking algorithms for selecting and counting for nuclei in the specific region of interest (ROI). The ROI was the 3D gel region between a PDMS post and the wall as seen in boxed area of Fig. 1b that was selected during confocal imaging and contained both the endothelial lining channel region as well as the collagen gel. ROIs were selected such that edge effects associated with PDMS walls and posts were avoided. The dimensions of the ROI were 250 mm6250 A 196 supplier mm6120 mm (height) and each microfluidic device contained total eight ROIs. While each ROIs were analyzed individually, the extravasation percentage was measured per device. As the tumor cells express GFP, cells with both green and blue signal were counted to track the number of tumor cells.Statistics Permeability of Endothelial MonolayerUpon formation of a complete endothelial monolayer by day 2, the diffusive permeability was measured with fluorescently-labeled dextrans in culture medium as shown in Fig. S1 (10 kDa cascade blue and 70 kDa MW Texas red, Invitrogen). The endothelial monolayers grown in our microfluidic system exhibited lower diffusive permeability values for the smaller molecular weight dextran confirm the presence of a size-selective endothelial barrier. To characterize changes in permeability upon extravasation, we used the 70 kDa dextran. Before introducing dextran into the device, the endothelium was first examined using a phase contrast microscope (Nikon, Tokyo, Japan) to confirm monolayer formation on both the top and the bottom of the channel by focusing at different heights. All medium in the device reservoirs was aspirated first and later re-filled with control medium in the side channels whereas.Librate. The tumor cell suspension medium in the channel was removed 1 hour later and all channels in the device were filled with endothelial cell culture medium. Control experiment with MCF-10A was done following the exact tumor cell seeding protocol. All cultures were kept in a humidified incubator, which was maintained at 37uC and 5 CO2.using OPENLAB 4.0.4 software. Images were later analyzed using MATLAB to calculate fluorescence intensity across the monolayer. To determine the 1655472 diffusional permeability, 25033180 we calculated the distribution of fluorescence intensity change as a function of distance perpendicular to the plane of the endothelial layer. A detailed procedure for measuring permeability has been described previously [24,35,36,37]. Briefly, we used the equation P = D [dC/ dx]/DCec where P is the diffusive permeability (cm/s), dC/dx is the gradient of the dextran concentration, DCec is the concentration difference across the monolayer, and D is diffusion coefficient of dextran.Immunofluorescent Staining and Image AcquisitionAll cells in the device were washed with Phosphate Buffered Saline (PBS) and later fixed with 4 paraformaldehyde for 15 min. After washing twice with PBS, cells were permeabilized with 0.1 Triton-X 100 solution for 5 min and blocked with 5 BSA solution for 5 h. VE-cadherin was labeled with rabbit polyclonal antibody (polyclonal; Alexis Biochemical) at 1:100 dilution and subsequently applied fluorescently-labeled secondary antibody. Cell nuclei were stained with DAPI (Invitrogen) at 1:1000 dilution. All images were obtained using a confocal microscope (Leica) and processed with IMARIS software.Metrics for ExtravasationQuantitative cell counting was performed after immunofluorescent staining. Confocal data were analyzed using IMARIS and its tracking algorithms for selecting and counting for nuclei in the specific region of interest (ROI). The ROI was the 3D gel region between a PDMS post and the wall as seen in boxed area of Fig. 1b that was selected during confocal imaging and contained both the endothelial lining channel region as well as the collagen gel. ROIs were selected such that edge effects associated with PDMS walls and posts were avoided. The dimensions of the ROI were 250 mm6250 mm6120 mm (height) and each microfluidic device contained total eight ROIs. While each ROIs were analyzed individually, the extravasation percentage was measured per device. As the tumor cells express GFP, cells with both green and blue signal were counted to track the number of tumor cells.Statistics Permeability of Endothelial MonolayerUpon formation of a complete endothelial monolayer by day 2, the diffusive permeability was measured with fluorescently-labeled dextrans in culture medium as shown in Fig. S1 (10 kDa cascade blue and 70 kDa MW Texas red, Invitrogen). The endothelial monolayers grown in our microfluidic system exhibited lower diffusive permeability values for the smaller molecular weight dextran confirm the presence of a size-selective endothelial barrier. To characterize changes in permeability upon extravasation, we used the 70 kDa dextran. Before introducing dextran into the device, the endothelium was first examined using a phase contrast microscope (Nikon, Tokyo, Japan) to confirm monolayer formation on both the top and the bottom of the channel by focusing at different heights. All medium in the device reservoirs was aspirated first and later re-filled with control medium in the side channels whereas.
