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Additional and updated supporting information including technical details may be found in the online version of this article.

FilenameFormatSizeDescription
cytoa22326-sup-0001-suppfig1A.tiff641KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by performing 1:2 serial dilutions. (A) Individual. fcs files were concatenated in order to visualize all titrations in a single pseudo-colour plot, where increasing Ab-concentrations are arranged along the x-axis. Titers used in OMIP-019 are highlighted in red. (B) To titrate TCR-DV1FITC and (C) TCR-DV2Ax594 for the detection of γδT-cell subsets, PBMC were co-labeled with TCR-GV9APC. Titers were selected to separate the positive population from negative cells, and maintain background staining at a minimum. (D) Specificity of CD1d-PBS57PE multimers was confirmed by co-staining with TCR-AV24FITC, TCR-BV11APC, and CD45RABV421. Unloaded CD1d multimers did not stain TCR-BV11+ cells. The TCR-AV24FITC vs. CD45RABV421 dot plot in the bottom right shows CD1d multimer+ TCR-BV11+ (green) overlaid onto classical T-cells (grey-black). Samples were gated on live T-cells (B-D).
cytoa22326-sup-0002-suppfig1BCD.tiff477KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by performing 1:2 serial dilutions. (A) Individual. fcs files were concatenated in order to visualize all titrations in a single pseudo-colour plot, where increasing Ab-concentrations are arranged along the x-axis. Titers used in OMIP-019 are highlighted in red. (B) To titrate TCR-DV1FITC and (C) TCR-DV2Ax594 for the detection of γδT-cell subsets, PBMC were co-labeled with TCR-GV9APC. Titers were selected to separate the positive population from negative cells, and maintain background staining at a minimum. (D) Specificity of CD1d-PBS57PE multimers was confirmed by co-staining with TCR-AV24FITC, TCR-BV11APC, and CD45RABV421. Unloaded CD1d multimers did not stain TCR-BV11+ cells. The TCR-AV24FITC vs. CD45RABV421 dot plot in the bottom right shows CD1d multimer+ TCR-BV11+ (green) overlaid onto classical T-cells (grey-black). Samples were gated on live T-cells (B-D).
cytoa22326-sup-0003-suppfig2.tiff26370KOnline Fig.2 Sample illustrations for choice of reagents. (A) Comparison of CD4QD605 and CD4BV605. (B) Performance of TCR-GV9PE and TCR-GV9APC were compared. Both reagents readily identify TCR-GV9hi cells, but the latter is better at also separating out the TCR-VG9dim cells. (C) Comparison of PE-Cy5.5-, PacBlu- and BV421-conjugates of anti-CD34 Ab. While it is not clear whether the PE-Cy5.5-conjugate stains haematopoietic stem cells (red broken-lined gate), the PacBlu-conjugate clearly identifies this population (red gate) and hints at a CD34dim population (green broken-lined gate), while the BV421-conjugate clearly identifies both these populations (red and green gates). (D) Staining intensity of CCR5PE-Cy7 and CCR5APC-Cy7. Dot plots show total live cells (A, C) or live T-cells (B, D) from healthy donor PBMC.
cytoa22326-sup-0004-suppfig3.tiff316KOnline Fig.3 Effect of incubation temperature on a subset of reagents used in OMIP-019. Staining performance comparison of CD1d-PBS57PE multimers, CCR5APC-Cy7, TCR-GV9APC, TCR-DV1FITC (A), TCR-DV2Ax594 (B), and CD34BV421 (C) after incubation at either room temperature or 37°C. Graphics show total live cells (A, C) or live TCR-DV9+ T-cells (B) from healthy donor PBMC.
cytoa22326-sup-0005-suppinfo.doc23KSupporting Information
cytoa22326-sup-0006-suppinfo.doc144K

Online Table 1 Instrument configuration.

Online Table 2 Commercial reagents used in OMIP-019.

Online Table 3 In-house conjugated reagents used in OMIP-019.

Online Table 4 Priority rating for reagents.

Online Table 5 Reagents tested but not included in final panel.

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