Gfr basement membrane matrix

Primary patient‐derived cancer organoids (PDCOs) were derived from radical prostatectomy specimen from patients with high‐risk localized prostate cancer as described before. Biopsy specimen was partially digested and propagated in hanging 50% Matrigel™ droplets (GFR Basement Membrane Matrix, BD Biosciences, Franklin Lake, NJ, USA) followed by transferring to Stacks wells in 50% Matrigel with PrEGM media (Lonza, Basel, Switzerland) supplemented with 0.1 μg EGF (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 10 μM Y‐27632 (Sigma‐Aldrich, Millipore Sigma, USA). PDCOs were treated with DMSO, 10 nM Docetaxel, or 100 μM Bicalutamide (Selleck Chemicals, Houston, TX, USA) for 72 h in PrEGM media supplemented with 10 μM Y‐27632 (Selleck Chemicals, Houston, TX, USA). Wells were rinsed three times with 1xPBS prior to staining with Image‐IT Dead™ Green, EpCAM Alexa Fluor 647, and Hoechst33342 (Supporting Information 2, Table S1) for 30 min at 37°C. Wells were then rinsed three times with PBS, fixed with 1% PFA (Cytofix, BD Biosciences) for 15 min followed by three washes with PBS. Wells were then mounted with SlowFade™ Gold Antifade Mounting Media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and imaged on a Nikon Yokogawa CSU‐W1 Spinning Disk Confocal Microscope equipped with Hamamatsu Orca Flash cameras. 20x images were then analyzed with NIS‐Elements software.