Antibodies used for <i>ex vivo</i> flow cytometry analysis.

a<p>Dump channel comprised of lineage markers to gate out non-relevant PBMC subsets.</p><p>n/a = not applicable.</p><p>BCR = B-cell receptor.</p

Antibodies used for post-culture flow cytometry analysis.

a<p>Dump channel comprised of lineage markers to gate out non-relevant PBMC subsets.</p><p>n/a = not applicable.</p><p>BCR = B-cell receptor.</p

Size and composition of the B-cell compartment <i>ex vivo</i> influences B-cell expansion during culture.

<p>Flow cytometry analysis was performed to determine proportions and subsequently calculate absolute numbers of (A) total CD19+ B-cells (blue dots) and (B) IgG+CD19+ B-cells (orange dots) <i>ex vivo</i> and post-culture. <i>Ex vivo</i> proportions of (C) total CD19+ B-cells (blue dots) within viable PBMCs were plotted against the fold change in their absolute numbers, and (D) IgG+ B-cells (orange dots) within total CD19+ B-cells were plotted against the fold change in their proportion within the B-cell compartment. (E) Proportions of IgG+CD19+ B-cells (orange dots) and (F) IgG−CD19+ B-cells (green dots) within viable PBMCs were plotted against the fold increase in their respective absolute numbers post-culture compared to <i>ex vivo</i>. Colored dots show cultures from all 269 stimulated samples (3–7 time points per volunteer), while black dots show the cultures from only the 62 baseline samples (1 for each individual volunteer). The black dashed line indicates the median fold change (with value), grey dotted lines represent the upper and lower limit of the interquartile range. Spearman r and p values are shown for analysis of baseline samples (black dots) from the 62 donors assessed.</p

Definition of B-cell subsets <i>ex vivo</i>.

<p>Definition of B-cell subsets <i>ex vivo</i>.</p

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-1

automatic total cell count, user correction thereof and parasitaemia scoring; blue crosses indicate where the user has added cells to the total cell-count, and green dots indicate where the user has subtracted cells. Yellow circles indicate cells that the user has deemed infected.<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-5

N arrows indicate unifected RBCs, and red arrows indicate infected RBCs. In this example the user has opted not to discriminate between the different stages of infected RBC. . A scored image of a human RBC culture experimentally infected with , captured at 600× magnification. In this example the user has chosen to specifically determine the number of trophozoites present (blue arrows), and infected cells at any other stage of development have simply been scored as 'infected RBC' (red arrows).<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films-0

N arrows indicate unifected RBCs, and red arrows indicate infected RBCs. In this example the user has opted not to discriminate between the different stages of infected RBC. . A scored image of a human RBC culture experimentally infected with , captured at 600× magnification. In this example the user has chosen to specifically determine the number of trophozoites present (blue arrows), and infected cells at any other stage of development have simply been scored as 'infected RBC' (red arrows).<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

The 'interphase' after automatic identification of individual cells and gridding of these ranked on various features potentially discriminating infected and uninfected cells

<p><b>Copyright information:</b></p><p>Taken from "Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films"</p><p>http://www.malariajournal.com/content/7/1/62</p><p>Malaria Journal 2008;7():62-62.</p><p>Published online 21 Apr 2008</p><p>PMCID:PMC2358916.</p><p></p

Induction of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells by malaria-infected RBCs is time and concentration-dependent.

<p>PBMC were cultured alone, with uninfected RBCs (nRBC) or iRBCs for 6 days at different iRBC∶PBMC ratios. Cells were analysed by flow cytometry for CD25 and Foxp3 expression in gated CD4⁺CD3⁺ lymphocytes. Dot plots show 1 representative of 12 donors examined (A). Comparative analysis of Foxp3 and isotype control staining on CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T cells in day 6 cultures revealed specific Foxp3 expression solely in the CD25⁺ population (B). Absolute numbers of different CD4 T cell populations for 4 donors (Mean+/−SEM) were calculated based on light microscopic cell counts and flow cytometry analysis on day 6 of culture of originally 2×10⁷ PBMCs co-cultured in the absence or presence of nRBCs and iRBCs (C). Data from 12 donors demonstrate iRBC concentration-dependent induction of Foxp3^hi and Foxp3^int CD4⁺CD25^hi T cells (D). A representative plot of 1 out of 7 donors shows the induction of these cells over a time course of 6 days (E).</p

Foxp3 induction requires soluble factors from monocyte-CD4 T cell interaction, but not necessarily direct contact of monocytes with converting T cells.

<p>To determine contact-dependency for converting T cells, T cells were separated from iRBC-co-cultured whole PBMC or T cells+monocytes through a 20 nm pore size transwell and analysed by flow cytometry on day 6. Bar graphs show data of 5 donors (Mean+/−SEM), analysed as fold change in the induction of CD25^hiFoxp3^hi or CD25^hiFoxp3^int CD4 T cells (A). Representative FACS plots for each condition are shown (B). Foxp3^hi∶Foxp3^int ratios were calculated for T cells in direct co-cultures as well as inside and outside the transwell (C). Foxp3^hi and Foxp3^int levels, as well as IL-2 and IL-10 levels in culture supernatants were monitored on day 2, 4, 5 and 6 and are shown for 1 out of 2 donors (D). To determine which T cell subpopulation has to interact with monocytes to induce Foxp3 expression in transwell-separated T cells, T cells that were added back to monocytes were depleted of CD4⁺, CD8⁺, or CD25⁺ cells prior to culture. Proportions of CD25^hiFoxp3^hi and CD25^hiFoxp3^int cells of CD4 T cells induced in the transwell-separated T cell compartment (on day 6) as well as IL-2 and IL-10 levels in the culture supernatant (on day 2 and 6, respectively) were measured and are shown as fold changes compared to whole T cells (E). Fold changes (black bars) were calculated by normalizing for each donor the percentage of each of the two cell types of CD4⁺ T cells or cytokine levels on the respective values obtained for monocytes+T cells directly cultured with iRBCs (A) or for cultures with whole T cells added back to monocytes within the transwell (E) (white bars). The T cell compartment analysed and depicted in each individual graph is highlighted in bold red. Experiments were set up with a 2∶1 iRBC∶PBMC ratio.</p