M30 apoptosense ck18 kit

Monospheroids and heterospheroids were seeded as described before [16 (link),19 (link)], and after one day, treated with 5 µM TGF-β receptor type I/II (TGFBR1/2) kinase inhibitor (LY2109761, Sigma-Aldrich, #SML2051) or different therapeutic compounds, including 50 µM gemcitabine (Sigma-Aldrich, #G6423), 1 µM paclitaxel (Sigma-Aldrich, #580555), 1 µM SN38 (active metabolite of irinotecan; Sigma-Aldrich, #H0165) or 5 µM pitavastatin (a HMG-CoA reductase inhibitor; Selleckchem, #S1759). Three days later, spheroids were collected for RNA extraction or apoptosis detection. M30 Apoptosense® CK18 Kit (Diapharma #P10011) was used according to the manufacturer's instructions to quantitatively detect apoptosis in epithelial cells and differences between treatment groups were assessed by paired t-test.

The organoids were labeled with Green Dye (Thermo Fisher, C7025), and organoid-primed T (opT) cells were added at a ratio of 1:10 (Tumor cell: T cell). Images were taken every 10 min starting at 2.0-hour co-culture for a total of 20 hours. For killing assay, opT cells were added to three-dimensional (3D) organoids at a ratio of 1:1 and incubated at 37°C for 24 hours, 48 hours, and 72 hours, respectively. Supernatants were collected and used for ELISA: M30 Apoptosense CK18 Kit (DiaPharma, P10011), interferon (IFN)-γ ELISA kit (Mabtech, 3420–1 H-20), and granzyme B ELISA kit (Mabtech, 3485–1 H-20).

Using fasting serum, circulating analytes [M30 Apoptosense® CK18 Kit (DiaPharma, OH) and M65 EpiDeath® ELISA (DiaPharma, OH)] were measured by ELISA. Circulating SHH concentrations were measured by ab100639 – Sonic Hedgehog Human ELISA Kit (Abcam, MA). All protocols were performed as per manufacturers’ instructions.