Cell Proliferation Assay

“IL33 WT” and “IL33 KO” cell lines were seeded at 3 × 10³ cells per well in DMEM + 10% FBS + 1% PS in four opaque-walled 96-well plates and left to adhere overnight. The following day, cell density was measured on 1 plate (day 0); the remaining plates were aspirated and washed with PBS, and cells were treated with low-serum DMEM or a 1:1 ratio of low-serum DMEM and 9805 “KRAS^G12D ON” tumor CM. Conditions without tumor CM were given doxycycline as a control. Plates were collected and read every 24 hours for 3 days. Proliferation was measured using CellTiter-Glo Luminescent Cell Viability Assay (#G7570, Promega) following the manufacturer’s instructions. Two wells per condition were averaged for each measurement.