Isolation of Colonic Epithelial Cells

Colonic epithelial cells were isolated as previously described^{35 , 36 (link)}. In brief, dissected colonic mucosa was cut into small pieces and incubated in magnesium-free Hank’s balanced salt solution (HBSS) containing 2 mmol/L EDTA and 2.5% heat-inactivated fetal bovine serum for 30 minutes with shaking at 37°C. Collected supernatants were centrifuged, resuspended in HBSS containing 1 mg/mL collagenase type 4 (17104019; Thermo Fisher Scientific), and incubated for 10 minutes at 37°C to further remove the mucus. The fraction was pelleted, resuspended in HBSS, passed through a 40-μm filter, and overlayered on 50% Percoll. Cells were centrifuged at 2000 rpm for 20 minutes at room temperature and viable colonic IECs were recovered from the interface layer.

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Levic D.S., Niedzwiecki D., Kandakatla A., Karlovich N.S., Juneja A., Park J., Stolarchuk C., Adams S., Willer J.R., Schaner M.R., Lian G., Beasley C., Marjoram L., Flynn A.D., Valentine J.F., Onken J.E., Sheikh S.Z., Davis E.E., Evason K.J., Garman K.S, & Bagnat M. (2024). TNF promoter hypomethylation is associated with mucosal inflammation in IBD and anti-TNF response. medRxiv.

Publication Preprint 2024

collagenase type 4

Corresponding Organization :

Other organizations : Duke University, Duke University Hospital, Duke Medical Center, University of North Carolina at Chapel Hill, University of Utah Health Care, Lurie Children's Hospital, University of Utah, Huntsman Cancer Institute

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Variable analysis

independent variables

Incubation in magnesium-free Hank's balanced salt solution (HBSS) containing 2 mmol/L EDTA and 2.5% heat-inactivated fetal bovine serum for 30 minutes with shaking at 37°C
Incubation in HBSS containing 1 mg/mL collagenase type 4 for 10 minutes at 37°C

dependent variables

Viable colonic IECs recovered from the interface layer after centrifugation on 50% Percoll

control variables

Dissected colonic mucosa
Passing the fraction through a 40-μm filter

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