T75 cm2 tissue culture flasks

Primary mixed glial cultures were isolated from neonatal (P2) Sprague–Dawley rats (Taconic Farms; RRID:RGD_1566440) as previously described (Osterhout et al., 1997). As these primary mixed glial cultures were obtained from entire litters of neonatal rat pups, the glial cultures were created from a mix of male and female animals. In brief, brains were removed and the cortices disassociated into a mixed glial suspension. Glial cultures were grown in T‐75 cm² tissue culture flasks (Corning) until the formation of a confluent astrocytic monolayer. OPCs were harvested using a mitotic shake off and purified by several differential plating techniques. OPCs were maintained in a defined, serum free media containing insulin, selenium, transferrin, and triiodothyronine, and supplemented with either B104 neuroblastoma conditioned media (B104‐CM, 30%v/v; RRID:CVCL_0154) or a mix of PDGF‐AA (10 ng/ml) and FGF2 (20 ng/ml) growth factors (PeproTech) to prevent OPC differentiation. The purity of each primary OPC preparation was assessed prior to utilization in experiments by staining with A2B5 and/or PDGFRα. The cultures were routinely at least 95% OPCs, with no contamination by microglia, and very few astrocytes.

HT-29 cells were cultured in T75 cm² tissue culture flasks (Corning) in RPMI GlutaMAX (Gibco) supplemented with 10% Foetal Bovine Serum (FBS Bovogen Biologicals) in 5% CO₂ at 37°C. Two days before infection HT-29 cells were seeded into 24 well tissue culture trays at a density of 2x10⁵ cells per well. The day before infection derivatives of EPEC were inoculated into LB broth and grown with shaking at 37°C overnight. On the day of infection, overnight cultures of EPEC were sub-cultured 1:75 in RPMI GlutaMAX (Gibco) and grown statically for 3 h at 37°C with 5% CO2. Where necessary, cells were induced with 1 mM IPTG (Sigma) for 30 min before infection. HT-29 cells were washed twice with PBS and infected with EPEC grown to an OD₆₀₀ of 0.06 for 3 h before being lysed for immunoblot analysis. Where required, after 3 hours of EPEC infection the HT-29 cells were treated for a further 2 hours with 100 μg/ml gentamycin to stop the infection, in combination with 20 ng/ml FasL before being lysed for immunoblot analysis.