2.7. Cell Culture Additives, Cholesterol-Depletion Assay, and Cholesterol Level Measuring

DEAE-dextran (Sigma, US), porcine bile extract (Sigma, US), and water-soluble cholesterol (Sigma, US) dissolved in DPBS or Milli-Q water (cholesterol only) were added to PIEs to achieve final concentrations of 50 µg/mL, 20 µg/mL, and 10 µg/mL, respectively. CMGF- containing the above additives or CMGF- alone (used as a negative control) were removed following 1 h of incubation at 37 °C, and the PIEs were washed twice with CMGF- medium and then infected with RVCs as described above.

Methyl-β-cyclodextrin (MβCD, Sigma, US), which has a high affinity for inclusion in the cell wall, competing with cholesterol and causing its cellular depletion, was used in the cholesterol-depletion assay. The cholesterol-depletion protocol has been described previously, and we used it with slight modifications [39]. Briefly, differentiated 3D PIEs were treated with 0 mM, 5 mM, or 20 mM MβCD at 37 °C for 1 h. For cholesterol replenishment, the PIEs were pretreated with 20 mM MβCD at 37 °C for 1 h, followed by washing with CMGF-, and then the cells were incubated with 1mg/mL (10 µg in total) of exogenous cholesterol for 1 h at 37 °C. To measure the PIE cholesterol levels, an Amplex™ Red Cholesterol Assay Kit (Invitrogen™) was used following the manufacturer’s instructions.