Csu w1 spinning disk confocal microscope

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.

The H358 lung cancer cell line expressing the KRAS G12C alteration was seeded on a collagen‐fibronectin matrix in Stacks and treated with 100 nM of adagrasib, a KRAS G12C small molecule inhibitor (Selleck Chemicals, Houston, TX, USA) or with DMSO for 48 h in serum‐free RPMI media. Cells were stained under live conditions with EpCAM Alexa Fluor 647 at 1:50 dilution, Image‐iT® Dead™ Green at 1:10 000 dilution, and Hoechst33342 for 60 min. Wells were rinsed with PBS, fixed with 1% PFA, washed with PBS, and mounted as described above. Cells were imaged directly in‐chip using a Nikon Yokogawa CSU‐W1 Spinning Disk Confocal Microscope equipped with Hamamatsu Orca Flash cameras, and the percentage of Image‐iT® Dead™‐positive cells were quantified using NIS‐Elements software. Spot detection function was used to identify individual cells and generate statistics on signal intensity values in each cell and for the sample as a whole. This experiment was performed twice with three technical replicates per experiment.