FIGURE 1.

Optimization of sublytic complement conditions. A, hemolytic activity testing lysis of ShEA by serum with or without 10 μg/ml OmCI (C5 blocker), titrated from 16% down to 0%. B, susceptibility of CT26 cells to C lysis; cells in a monolayer were loaded with calcein AM then exposed to serum for 1h at 37 °C. Lysis was calculated from the release of calcein into the supernatant and expressed as the percentage of the total entrapped calcein obtained by detergent lysis of the cells. Results are the means of four separate experiments ± S.E. C and D, expression analysis of OPN in CT26 cells in response to exposure to sublytic C for 1, 6, and 12 h. CT26 cells were exposed for 1 (C) and 6 and 12 (D) hours to 5% serum treated with or without a MAC-blocking dose of OmCI and OPN gene expression analyzed by qPCR. Expression was calculated as % of untreated control. Results are means of three determinations ± S.E. (*, p < 0.05; **, p < 0.01).