Imprime binding to neutrophils and monocytes in whole blood is complement-dependent.

<p>(A) Binding of increasing concentration of Imprime (0, 0.1, 1, 10, and 25 μg/mL) to human neutrophils and monocytes was measured by flow cytometry after incubation of WB with Imprime or vehicle at 37°C for 30 mins. (B) Imprime binding in the absence of serum (serum removed), heat-inactivated serum (HI serum), presence of anti-C3 peptide, compstatin (100 μM) or α-CR1 mAb (10 μg/mL) (right column in each respective row) are compared to the binding to vehicle or Imprime of untreated WB (left and middle column in each respective row). For compstatin and α-CR1 mAb treatment, WB was pre-treated with the blocking agents at 4°C for 30 mins prior to the incubation with 10 μg/mL Imprime at 37°C for 30 mins. (C) The role of classical pathway in Imprime binding to neutrophils and monocytes was evaluated by blocking with the anti-C1q mAb (50 μg/mL) at 4°C for 30 mins prior to the incubation with Imprime or vehicle. (D) Imprime binding to enriched human neutrophils and monocytes in 20% serum, 20% C1q-depleted serum (C1q-dep serum), or 20% C1q-depleted serum replenished with 100 μg/mL purified human C1q protein (C1q-dep serum + C1q protein) was determined by flow cytometry. The MFI and percentage of BfD IV positive cells are indicated on the contour plots. Data shown for each part are representative of 3–5 independent experiments performed with different donors.