Effect of anti-P2 mAb E2G12 on <i>Plasmodium</i> cells.

<p>Synchronized <i>P. falciparum</i> cells were treated with no antibody (control); SP2/O: medium from a non-Ig-secreting cell line; and various mAbs (anti-PfP0: E5F4; anti-β actin and anti-PfP2: E2G12) each of the same isotype IgG1, used at 1 mg/ml final concentration. The distribution of the SN/DN/TN/MN stages of the IEs were determined through DAPI count. SN: IE with single nucleus; DN: IE with di-nuclei; TN: IE with tri-nuclei; MN: IE with >3 nuclei (<b>A</b>) Various antibodies were added at 12 hrs PMI and the population was scored at 24 and 48 hrs. (<b>B–C</b>) Synchronized <i>P. falciparum</i> cells were treated with E2G12 mAb for 24 hrs starting from 12 to 36 hrs PMI. At 36 hrs the arrested cells were washed and split into two flasks and cultured for further 20 hrs with and without E2G12 (antibody continued and removed, respectively). The population of IE were scored using DAPI at 0, 2, 6, 10, 20 hrs post washing; corresponding to 36, 38, 42, 46 and 56 hrs PMI. *P<0.05, **P<0.01, ***P<0.001. Significance of each time point was calculated with respect to the immediate previous time point; n = 5. Standard deviation has been represented as an error bar. (<b>D, E</b>) Permiabilized IFA of cells at 0, 2, 6 and 8 hrs post washing, corresponding to 36, 38, 42 and 44 hrs PMI using DAPI and anti-P2 E2G12 mAb. (D): after E2G12 mAb was washed off and (E) with E2G12 mAb treatment continued. Anti-tubulin antibodies were also used to stain the microtubule organization center (MTOC) of the arrested cells. (<b>F, G</b>) SIFA for actin staining of cells that were treated for 24 hrs of E2G12, and then with E2G12 washed off (F) and E2G12 continued (G). No actin staining was observed showing that treatment with E2G12 did not cause membrane permeabilization. Scale bar indicates 2 µm. (<b>H, I</b>) Flow cytometric analysis of DAPI and P2 staining of E2G12 treated cells (E2G12 added at 12 hrs PMI) under SIFA conditions. Staining was done in control cells (no antibody), and in cells with E2G12 continued and after E2G12 was washed at 36 hrs PMI. For analysis, total 6 million cells were counted. Representative data for the dot-plot and the P2 fluorescence intensity data are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858.s008" target="_blank">Figures S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002858#ppat.1002858.s009" target="_blank">S9</a>, The intensity of DAPI values shown in panel H are in comparison with the unstained cells. The <i>p</i> value depicts a statistical comparison with the control (no antibody) value for each time point. * <i>P</i><0.05, n = 3.