Clone g8

Apoptosis was analysed by fluorescence microscopy using an in situ cell death detection kit (Roche). Caspase activity was measured by incubation of 25 μg tissue homogenate with 50 μM acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-amc) (Peptide Institute, Osaka, Japan) in 150 μl cell-free system buffer (10 mM HEPES–NaOH pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH₂PO₄, 0.5 mM EGTA, 2 mM MgCl₂, 5 mM pyruvate, 0.1 mM PMSF, 1 mM dithiothreitol). The release of fluorescent 7-amino-4-methylcoumarin was measured for 50 min at 2-min intervals by fluorospectrometry at 360 nm excitation and 480 nm emission wavelength, using a Cytofluor device (PerSeptive Biosystems, Cambridge, MA). The maximal rate of increase in fluorescence was calculated (Δfluorescence/min). Lungs were prepared as described for flow cytometric analysis and cells were stained with antibodies directed against CD326 (EpCAM, eBioscience clone G8.8), CD45 and CD31 (eBioscience, clone 390). Prior to flow cytometric analysis cells were washed and incubated with Annexin V and propidium iodide in Annexin V binding buffer according to manufacturer’s instructions (BD Biosciences).

Colon crypt single cell suspensions were DNAse treated for 5 min (Sigma-Aldrich #4716728001). Following a wash step, cells were incubated for 30 min in FACS buffer (phosphate buffered saline with 3% fetal bovine serum + 10 µM Rock inhibitor (Y-27632 AdipoGen Life Sciences from Fisher #501146540)) with the following pre-conjugated validated flow antibodies: CD45-BV510 (1:200, Clone 30-F11; BD Biosciences #563891), CD31-BV510 (1:200, Clone MEC 13.3; BD Biosciences #563089), CD326-eFluor450 (1:100, Clone G8.8; eBioscience #48-5791-82), CD44-PerCP-Cy5.5 (1:100, Clone IM7; Thermo Fisher #A26013), CD24-PECy7 (1:200, Clone M1/69; eBioscience #25-0242-82), and CD117-APC-Cy7 (1:100, Clone 2B8; Thermo Fisher #A15423). Following wash steps, cells were resuspended in FACS buffer and Live/Dead Aqua (Thermo Fisher # L34957). An alternative CD45-APC (1:200, Clone 30-F11; BD Biosciences #561018) antibody was used in the Supplementary Fig. 7f where specified.