Orca flash 4.0 v2 digital cmos camera

Patient-derived GBM cell lines, HK217 (proneural), HK301 (proneural) and HK280 (mesenchymal) were used for encapsulation. Sizes of GBM spheroids were standardized by seeding approximately 600K cells per well into Aggrewell™ well plates (Stemcell Technologies) one day prior to encapsulation. The following day, spheroids were harvested from the wells, centrifuged briefly (200×G, 1 min) and resuspended in the hydrogel precursor solution. Spheroid-laden hydrogels were formed as described in the above hydrogel fabrication section. Cell migration was observed periodically (Imaged at Day 1,3,6 and 9) by acquiring phase contrast images on a Zeiss Axio.Z1 Observer microscope with a Hamamatsu Orca Flash 4.0 V2 Digital CMOS Camera and Zeiss ZEN 2 (Blue Edition) software. Quantitation is described separately. At the end of experimental period, hydrogels were fixed using 4% paraformaldehyde (PFA) and stained with Hoescht (nuclei) and Cell Mask™ (cell membrane). These gels were imaged using a Leica SP5 confocal microscope. To block ITGαV-IBSP interaction, GBM spheroids were incubated with 10 μg/mL anti-ITGαV antibody a day prior to encapsulation. In addition, after the encapsulation, hydrogels were incubated in GBM media containing 10 μg/mL of the same antibody over the course of the experiment.

We acquired digital, fluorescence images of mIHC slides using Zeiss Axio Scan.Z1 with Zeiss 20X (0.8NA, M27) Plan-Apochromat objective, Hamamatsu ORCA-Flash 4.0 V2 Digital CMOS camera (16-bit; 0.325 μm/pixel resolution), and Zeiss Colibri.7 LED Light Source. We used the following filter specifications: DAPI cube (Zeiss Filter Set 02), FITC cube (Zeiss Filter Set 38 HE), Cy3 cube (Chroma Technology Corp 49004 ET CY3/R), Cy5 cube (Chrome Technology Corp 49006 ET CY5), Cy7 cube (Chroma Technology Corp 49007 ET CY7). After image acquisition, we converted images to 8-bit JPEG2000 format (100% quality).