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Enteroid monolayers were grown as described above for 5–7 days to 100% confluence. The cells were washed once with sterile PBS at 37 °C and once with culture medium without antibiotics upon inoculation with ETEC. The wild type ETEC strain GIS26 and an F4 deficient mutant strain GIS26∆faeG were grown overnight at 37 °C in 5 mL BHI medium while shaking at 180 rpm [29]. Culture density was measured at OD660 and verified by overnight plating on BHI agar plates and colony count. After washing with sterile cold PBS, the bacteria were added to the monolayers in culture medium at a multiplicity of infection (MOI) of 10 in duplicate and incubated for 2 h. Next, the excess bacteria were removed by washing the cultures three times with PBS, after which the cells were detached using 0.25% trypsin in PBS for 30 min at 37 °C. Subsequently, a serial dilution was plated on BHI agar and after overnight incubation at 37 °C the number of colony forming units (CFU) were counted.
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