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. 2019 May 8;2:172. doi: 10.1038/s42003-019-0429-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A quantitative framework for assessing the number of PfEMP1 molecules per knob. a Single-molecule detection events are shown as a function of the fluorescence cluster size for the G6 strain grown in HbAA (dark red dots) and HbAS (dark blue dots) erythrocytes²⁵. b Calibration curve showing the blinking distribution curve of single mEOS2 molecules immobilized on a glass surface²⁵. A single exponential decay function was fitted to the data points. c Number of PfEMP1 molecules per knob for G6 at the trophozoite stage grown in HbAA and HbAS erythrocytes, as determined by PALM. A total of 771 and 638 knobs, respectively, were analyzed²⁵. A box plot analysis is overlaid over the individual data points, with the median and the 25 and 75% quartile ranges being shown (also in the following subfigures). Statistical significance was assessed using the Mann–Whitney rank sum test (also in the following subfigure). d Absolute number of surface-presented PfEMP1 molecules (VAR2CSA variant) per single erythrocyte infected with the parental FCR3^CSA strain as a function the time post invasion, as determined by quantitative FACS analysis using the Zenon-labeled monoclonal antibody PAM 8.1 directed against VAR2CSA. The mean ± SEM are shown for n = 4 or more independent biological samples / blood donors²⁵. Red and blue circles, FCR3^CSA grown in HbAA and HbAS erythrocytes respectively. The insert shows a representative calibration curve to convert fluorescence signals into absolute molecule numbers. e Knob density and knob diameter for HbAA and HbAS erythrocytes infected with FCR3^CSA (trophozoites), as determined by scanning electron microscopy²⁵. Data are derived from x = three independent biological samples / blood donors. f Number of PfEMP1 molecules per knob for FCR3^CSA grown in HbAA (red) and HbAS (blue) erythrocytes, as determined by quantitative FACS analysis and taking into account cell surface area and knob density²⁵. Each data point corresponds to an independent biological sample based on a different blood donor. The following anti-VAR2CSA monoclonal antibodies were used: PAM 8.1 (red circles), PAM 3.1 (red triangle), PAM 4.7 (red square)