Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were obtained from commercial vendors, stained as described in the online section of the manuscript and acquired on a BD FACSDiscover™ S8. The optical configuration of the instrument is described in - . Additional gating and staining controls are shown in - . Pre-gating of plots is annotated in the figure or indicated by dotted black arrows. For some plots different donors are shown for clarity. The gating strategy has been devised in such a way that the staining pattern for every marker in the panel can be shown at least once on a single A4 page. The raw data has been deposited on Flowrepository with the identifier FR-FCM-Z73V. Abbreviations: BUV: Brilliant Ultraviolet; BV: Brilliant Violet; BB: Brilliant Blue; RB: RealBlue; AF: AlexaFluor; PE: Phycoerythrin; APC: Allophycocyanin; Qdot: Quantum Dot; NIR: Near-Infrared; (A) Gating strategy for CD45+ live cells, monocytes, B cells and γδ and αβ T cells. (B) Overview of the 50 targets analyzed with this experiment. Some of the markers can be used for phenotyping multiple immune cell lineages. (C) Representative plots for the main phenotyping markers in the B cell lineage (IgG, IgD, IgM and CD24). (D) Gating strategy to delineate invariant NKT cells, MAIT cells, CD4 + and CD8 + T cells, as well as CD4 + regulatory T cells (Tregs). (E) Representative plots for CD69, CD103, CD57 and PD-1 expression on non-naïve CD8 + cytotoxic T cells. (F) Expression pattern for CD39, CXCR3, CCR4, CD45RO and ICOS on the CD4 + Treg population. (G) Histogram overlays for the expression pattern of BTLA, CD27, CD28, CD38, TIGIT and KLRG1 on NK cells (grey), MAIT cells (orange), CD4 + Tregs (red), CD4 + non Tregs (purple), CD8 + naïve T cells (green) and CD8 + non-naïve T cells (blue). Dotted red lines indicate positivity cut-offs. (H) Gating strategy for NK cells and NK cell subsets based on CD56, CD161, CD16 and Nkp46. (I) Gating strategy for Basophils (CD123 + FcER1 + HLA-DR - ), plasmacytoid DCs (CD303 + HLA-DR + ), pan conventional DCs (CD11c + HLA-DR + ), and the cDC1 (CD141 + ) and cDC2 (FcER1 + ) subsets. (J) Histogram overlays for the expression pattern of CD86, CD40, CD11b, CD1c and CD163 on B cells (grey), CD8 + naïve T cells (green, negative control), CD14 + monocytes (purple), CD16 + monocytes (yellow), CD141 + cDC1s (red) and FcER1 + cDC2s (blue). Dotted red lines indicate positivity cut-offs. (K) Gating strategy for Lin - CD2 + CD127 + innate lymphoid cells (ILCs). (L) A selection of fluorescence-minus-one (FMO) controls for the indicated markers: PD-1 and BTLA on CD8 + T cells, ICOS and CCR4 on Tregs, and CD86 and CD1c on Lin-cells as indicated. Dotted red lines indicate positivity cut-offs. Note that there is no or only negligible spreading error (SE) present. Additional FMO and gating controls are shown in .

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, Marker, Expressing, Negative Control, Selection, Fluorescence

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were stained with anti-human CD4 antibodies conjugated to the indicated fluorochromes and acquired on a BD FACSDiscover™ S8 at standard settings. Fluorochrome similarity index was calculated as the cosine of the angle of the fluorochrome vector across the entire detector space.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, Plasmid Preparation

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were stained with anti-CD4 antibodies conjugated to the indicated fluorochromes, and acquired on a BD FACSDiscover™ S8 at standard settings. The individual single-stains were unmixed either by themselves (with Autofluorescence, blue bars) or in the context of the full 50-color unmixing matrix (orange bars). Fluorochromes are ordered based on descending stain index (SI) using the single-color unmixing. Stain index was calculated using the standard formula .

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were obtained from commercial vendors, stained with the full 50-color (50c) or the trimmed-down 45-color (45c) panel and acquired on a BD FACSDiscover™ S8. (A) Additional fluorescence-minus-one (FMO) controls for the indicated markers: CD38, CD25, CXCR3 and KLRG1. Pre-gating is indicated above the plots. Dotted red lines indicate positivity cut-offs. (B) Histogram overlays for the indicated markers on B cells (grey), monocytes (green), cDCs (blue), NK cells (orange) and pan T cells (red). (C) Plots from the full 50c stain (top panel) and trimmed-down 45c panel (lower panel) lacking the indicated reagents. For the plots in panel (C) OLS unmixing data is shown. Dotted red lines indicate positivity cut-offs. Pregating is indicated above the plots.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were obtained from commercial vendors, stained with the 50-color panel and sorted for four different populations on a BD FACSDiscover™ S8. Instrument setup for cell sorting followed best practices and the instructions from the manufacturer. The instrument was operated at a sheath pressure of 35psi with an 85um nozzle. (A) Gating strategy to delineate the main immune cell subsets and all populations that were planned for sorting. Sorted populations are marked with colored boxes. (B) Re-analysis of the indicated populations post sorting. Color-code referring to the gating tree.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, FACS

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: Data from the full 50-color panel acquired on the BD FACSDiscover™ S8 was gated on CD45+ live events, subsampled to 500.000 events and subjected to UMAP and FlowSOM calculation, with 30 metaclusters. (A) FlowSOM metaclusters overlaid onto a UMAP plot with manual annotation of populations. (B) Heatmap overview of marker expression (scaled) for all FlowSOM clusters. (C) Histogram representation of the indicated markers across all FlowSOM clusters. Dotted red line indicates manual positivity cut-offs. Note the consistent and tight distribution of negative peaks across almost all clusters.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Marker, Expressing

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: (A) Spectral similarity indices and spectral matrix condition numbers (CN) as measured on the BD FACSDiscover™ S8 for a subset of violet-excited fluorochromes in two nearly identical versions of our 50-color panel, using either eFluor 506 (top) or AmCyan as in the final panel (bottom). Both versions have identical CN values and comparable similarity indices, with the closest spectral neighbors for both dyes being BV480 and BV510 (having similarities of 0.82 and 0.83 respectively for eFluor 506, or 0.87 and 0.74 for AmCyan). (B) Analysis of unmixing spreading error (shown here with overlaid plots of unmixed anti-CD4 single stain samples) in the context of both full panels reveals substantially worse unmixed spread and resulting loss of resolution for eFluor 506 compared to AmCyan. Based on this analysis, we chose to proceed with AmCyan in the final panel. (C) Comparison of CN for the final 50-color and 45-color panels reported in this manuscript (orange) and three previously published OMIPs (blue). CN calculations were based on spectral signatures measured on the BD FACSDiscover™ S8. Spectral signature data for Live/Dead Fixability Blue (Thermo Fisher) was not available, so calculations were performed using the spectrum of the nearly identical BD Fixable Viability Stain 440UV instead.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, Comparison

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were stained with anti-human CD4 antibodies conjugated to the indicated fluorochromes and acquired on a BD FACSDiscover™ S8 at standard settings. Fluorochrome spectral signatures (normalized emission spectra) are shown as line plots grouped by primary laser (top) and as a heatmap (bottom).

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were obtained from commercial vendors, stained with the full 50-color panel and acquired on a BD FACSDiscover™ S8. Plots depict either all CD45+ live events or CD45+ non monocyte events, as indicated above plots. These fluorochrome pairs showed the highest similarity indices in our fluorochrome selection (see ), but still resolve well with relatively low spreading error (SE).

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining, Selection

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: PBMCs were obtained from commercial vendors, stained with the full 50-color (50c) panel and acquired on a BD FACSDiscover™ S8. Three different donors are shown. (A) 2D plots for CD57 and PD-1 on the CD8+ non-naïve T cell population (left side, gated as in main ) and histogram plots for the five indicated markers across the listed T cell populations. (B) 2D plots for CD141 and FcER1 on the CD11c+ HLA-DR+ cDC population (left side, gated as in main ) and histogram plots for the five indicated markers across the listed B cell, monocyte and DC populations. Red dotted lines show positivity cut-offs.

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining

Journal: bioRxiv

Article Title: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

doi: 10.1101/2023.12.14.571745

Figure Lengend Snippet: Reagent-matched single-stain controls were prepared using both PBMCs and BD Comp Beads and acquired on a BD FACSDiscover™ S8 at standard settings. Normalized spectra match closely between beads and cells for most fluorochromes (PE-Cy5 shown as an example, top panel), but can differ significantly for other fluorochromes (BV711 and BUV805 shown as examples, center and bottom panels, see Delta Cell-Bead).

Article Snippet: The optical setup of the Sony ID7000™ had the same excitation laser wavelengths as the BD FACSDiscover™ S8 and in addition a near UV laser (320nm) and Near-Infrared laser (808nm).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Development of a customizable mouse backbone spectral flow cytometry panel to delineate immune cell populations in normal and tumor tissues

doi: 10.3389/fimmu.2024.1374943

Figure Lengend Snippet: The performance of the backbone is reproducible across different spectral flow cytometers. (A) Manual gating strategy showing the main immune populations. Wildtype spleen cells were stained with the backbone panel and acquired on Cytek Aurora (in pink –top), Sony ID 7000 (in blue –middle), and BD S6 SE (in green –bottom). (B) Comparison of the frequency of the main immune populations of live CD45 + cells shows no significant differences across the three instruments (n=3 mice/instrument). Data are ± mean s.e.m. Statistical analysis was performed using two-way ANOVA with Geisser-Greenhouse correction, followed by Tukey’s multiple comparisons tests to compare the mean values of the immune cell population percentages between the two instruments (all p values were > 0.1.).

Article Snippet: Finally, our backbone panel performance is consistent across all the major spectral flow cytometer systems currently available (i.e., Cytek Aurora, Sony ID 7000, BD FACSymphony S6 SE), making it a dependable and widely applicable tool for researchers to study immune cell populations in murine cancer models.

Techniques: Staining, Comparison

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Principles of Advanced Flow Cytometry: A Practical Guide

doi: 10.1007/978-1-0716-2740-2_5

Figure Lengend Snippet: Comparison of high-dimensional flow cytometry data obtained on conventional and spectra flow analyzer. Two-by-two contour plots of a 19-color high-dimensional flow cytometry panel staining of mouse splenocytes for a variety of T-cell markers. The same sample was acquired on a 25-color BD FACSymphony A5 conventional and a 110-detector Sony ID7000 spectral flow analyzer. Both instruments are equipped with the same type of five lasers, though laser powers are different. Major cell types, subpopulations, and their percentage proportions are indicated. Not all parameters are displayed. Analysis was performed in FlowJo v10.8.1

Article Snippet: Currently Sony Biotechnology’s ID7000 and Cytek Biosciences’ Aurora models are capable of more than 20-color spectral analysis, and BD Biosciences are also beginning to offer spectral upgrade on their FACSymphony A5 models.

Techniques: Comparison, Flow Cytometry, Staining