The 3D7 P. falciparum clone and one Ghanaian patient isolate (BM057) were cultured in vitro (26 (link)) and were selected for DC4 PfEMP1 IE surface expression by repeated Ab selection as described previously (12 (link)). The identity of the isolates was routinely verified by genotyping as described previously (27 (link)), and Mycoplasma infection was regularly excluded using the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer’s instructions.
P. falciparum IE were DNA-labeled with ethidium bromide and surface-labeled with mouse antisera obtained from the immunized mouse used for hybridoma production (15 μl serum/well), 24E9 mAb (100 μg/ml), or 24E9 Fab fragments (100 μg/ml). Whole Abs were labeled using an FITC-conjugated secondary anti-mouse IgG (1:100; Vector Labs), and an anti-mouse F(ab′)2 IgG (1:100; Jackson Immunoresearch) was used to detect Fab fragments. FITC fluorescence data from ethidium bromide+ cells were collected on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and analyzed in WinList version 6.0 (Verity Software House).
P. falciparum IE were DNA-labeled with ethidium bromide and surface-labeled with mouse antisera obtained from the immunized mouse used for hybridoma production (15 μl serum/well), 24E9 mAb (100 μg/ml), or 24E9 Fab fragments (100 μg/ml). Whole Abs were labeled using an FITC-conjugated secondary anti-mouse IgG (1:100; Vector Labs), and an anti-mouse F(ab′)2 IgG (1:100; Jackson Immunoresearch) was used to detect Fab fragments. FITC fluorescence data from ethidium bromide+ cells were collected on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter) and analyzed in WinList version 6.0 (Verity Software House).