Insertion of 7-ketocholesterol and cholesterol in the plasma membrane

To generate membrane disorder lymph node cells isolated from DO11 TCR transgenic mice were centrifuged and resuspended in Opti-MEM Media (Invitrogen-Life Sciences, Grand Island, NY), at 1 × 10⁶/ml cell concentration before incubating with 7-ketocholesterol (7-KC) Methyl-beta-cyclodextrin (MβCD) (Sigma Aldrich, MO, USA) complexes for 10 min at room temperature as previously described²⁶. The 7-KC and MβCD complexes were generated by mixing each of the ethanol soluble 7-KC at 70 µM, 35 µM and 17.5 µM solutions with water soluble 0.3 mM MβCD. Cholesterol-MβCD complexes were generated similarly. Insertion of oxysterols (and cholesterol) into the plasma membrane is facilitated by MβCD^27,28. Incubation with 7-KC- MβCD (and cholesterol-MβCD) complexes, was carried out in Opti-MEM to reduce the exposure to serum cholesterol. To restore membrane order, cholesterol (35 µM & 17.5 μM) MβCD (0.3 mM) (Sigma Aldrich, MO, USA) complexes were added to cells following (soon after) the addition of 7-KC-MβCD complexes and incubated for 10 min at room temperature.