Shiga toxin glycosphingolipid receptors of Vero-B4 kidney epithelial cells and their membrane microdomain lipid environment

Membrane assembly of Shiga toxin glycosphingolipid receptors and toxin refractiveness of MDCK II epithelial cells

“…Cell viability was determined using the crystal violet assay as previously described (46,74). In short, MDCK II cells were grown to confluence in tissue culture flasks (Greiner Bio-One), trypsinized and seeded in 100 µl volumes in 96-well tissue culture plates (Corning Inc., Corning, NY, USA) (initial cell seeding density of 2 x 10 3 cells/well).…”

“…Remaining adherent cells were fixed with formalin, stained with crystal violet and cell survival was quantified photometrically as previously described (46,74 …”

“…The isolation of neutral GSLs from lipid extracts of total cells was performed following established protocols previously published (46,75,76 (46,75,76).…”

“…A phospholipid reference preparation (R3) comprising sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS) was employed as described in several previous publications (46,74,75,82). Cholesterol was purchased from Sigma Aldrich (cat.…”

“…Classical DRM and nonDRM fractions were prepared from sucrose density gradients according to the commonly used procedure described by Brown and Rose (67) with minor modifications as previously published (46,74,75,82 …”