Effect of culture in SFM on erythrocytes.
Erythrocytes display reduced viability and increased cell-surface PS exposure upon culture in SFM. (A) Freshly isolated erythrocytes were cultured at 5 × 106 cells per milliliter in medium, either alone or supplemented with 10% FBS, 10% PPP, or 10% human serum. Cell number was determined for triplicate cultures at time 0 and every 24 hours or 48 hours thereafter. Data are presented as mean cell number ± SD. (B) PS translocation was determined by annexin-V FITC binding at 0, 4, and 6 days following initial culture of the cells in SFM. The thin line represents erythrocyte autofluorescence in the absence in annexin-V FITC. Bound annexin-V FITC fluorescence is represented in bold. Erythrocyte membrane integrity at 0, 4, and 6 days of culture in SFM was determined by incubating the cells with phalloidin FITC. As a positive control for phalloidin FITC uptake by erythrocytes, 5 × 106 cells per milliliter were fixed with 1% phosphonoformatic acid and permeabilized with methanol prior to addition of phalloidin FITC. The thin line represents erythrocyte autofluorescence in the absence of phalloidin FITC. Phalloidin FITC fluorescence is represented in bold.
