Colonoid Infection Assay with EAEC

Human colonoid line 75C was established from de-identified biopsy tissue from a healthy subject provided informed consent at Johns Hopkins University. All methods were carried out in accordance with guidelines and regulations approved by the Johns Hopkins University Institutional Review Board (IRB# NA_00038329). Colonoids were maintained as previously described [28 (link)]. Colonoid monolayers were cultured in a 96-well plate pre-coated with human collagen IV (30 μg/ml; Sigma-Aldrich, USA) until confluency and then differentiated for 3–4 days. EAEC strains were grown for 16–18 hours without shaking in DMEM-high glucose (Gibco). Approximately 1 x 10⁶ CFUs were added to each well. After a 3 hour incubation, the monolayers were washed three times with PBS, lysed with 1% Triton X-100/PBS, and adherent bacteria were enumerated by dilution and plating on Luria agar.