differentiation 36  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures

doi: 10.3389/fimmu.2026.1959895

Figure Lengend Snippet: Comparison of PaCMAP-based and traditional 2D biaxial gating consistency for leukocyte subset identification. CD-CHEX–stabilised blood from two donors (different lots; CD1 and CD2) was gated across up to four experimental timepoints by three independent expert operators using either traditional 2D hierarchical biaxial gating (purple) or PaCMAP dimensionality-reduction-based gating (green). Gating purity was assessed against UMAP-defined reference gates drawn by a single expert. (A) Longitudinal trajectories of correct-assignment percentages (% cells landing in the corresponding UMAP reference gate) across timepoints (Vo1 to Vo4), faceted by population (columns); the two donors are distinguished within each facet by point shape (circle, triangle) and mean-line style (solid, long-dashed). Each bold line links the per-donor mean at each timepoint (filled points, colored by method); dotted light lines are individual operator trajectories (largely overlapping and difficult to discern for PaCMAP). The y-axis is pseudo-log-scaled to resolve values near 100% (using scales::pseudo_log_trans(sigma = 0.1) in R). (B) Boxplots of correct-assignment percentages per population; boxes show the interquartile range (IQR), whiskers extend to 1.5 × IQR, white diamonds mark the group mean, and dots are individual operator × timepoint observations, colored by method and shaped by donor. (C) Boxplots of contamination percentages (% cells from a gated population landing in a non-corresponding UMAP reference gate) faceted by UMAP gated population, on a pseudo-log axis to accommodate the many zero values; boxes and dots as in (B) , with donor shown by point shape. (D) Summary of Wilcoxon rank-sum comparisons (Benjamini–Hochberg-adjusted) between methods across six metrics: correct-assignment temporal stability (SD), correct-assignment inter-rater ICC, mean contamination, proportion of zero-contamination events, contamination temporal stability (SD) and contamination inter-rater ICC; columns give each method’s group mean, the adjusted p-value and the direction; cell color indicates the significant direction (green, PaCMAP superior; orange, 2D superior; white, p ≥ 0.05). ICC (intraclass correlation coefficient) was computed with a two-way model, absolute agreement, single unit (irr::icc); groups with undefined between-operator variance were excluded as degenerate (fewer than two timepoints or three raters, or near-zero overall variance [SD < 0.01]; groups with identical values across raters were treated as perfect agreement), leaving n = 15 (PaCMAP) and n = 16 (2D) for correct assignment. Temporal-stability SD, mean contamination and the zero-contamination proportion were computed per operator and then compared between methods, isolating within-operator consistency; ICC uses the three operators as raters and therefore quantifies between-operator agreement.

Article Snippet: CD-CHEX Plus stabilized human blood (213323, lots 32680071, 41550071, 50690071 and 53210071, Streck) was used as a biological-invariant control.

Techniques: Comparison, Stability, Immunocytochemistry

Journal: Frontiers in Immunology

Article Title: An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures

doi: 10.3389/fimmu.2026.1959895

Figure Lengend Snippet: Pre-analytical handling effects on leukocyte composition and two-tier normalization. Whole blood from healthy donors was processed under varying pre-analytical conditions (n = 3 replicates per condition) and immunophenotyped by flow cytometry; populations are expressed as a percentage of total leukocytes. (A–D) Mean leukocyte subset composition (stacked bars: neutrophils, monocytes/macrophages/dendritic cells, eosinophils, B cells, T cells) per condition, with the five-population Bray–Curtis PERMANOVA result above each panel: (A) refrigerated storage at 4°C for 1, 2 or 3 days (Day 1 fresh reference; samples were pooled then split from donor 1, 4, and 6); (B) cryopreservation in CryoStor CS10 or DMSO for 15 days versus fresh (samples were split from donor 4); (C) chemical fixation analyzed at Day 1 or Day 5 versus unfixed Day 1 (samples were split from donor 4); (D) one-week-stored versus freshly prepared antibody cocktail (samples were split from donor 4). Companion data of numerical mean values in . (E–H) Two sequential leukocyte count normalization tiers were compared against raw counts for longitudinal stability. Tier 1 rescaled within-sample (to CD45 + % [%CD45], or to cells/mL by counting beads [bead] or volume [vol]); Tier 2 added a CD-CHEX-anchored cross-batch correction to remove run-to-run drift (schemes detailed in ): (E) Lineage populations of CD-CHEX, including eight major lineage populations (as described in ), were assessed for count stability over time (y-axis, Bray–Curtis dissimilarity to the first timepoint per tier, x-axis, acquisition month; lower and flatter = more stable). Each line is one normalization scheme (color) for one held-out CD-CHEX lot (line style and point shape distinguish the two lots, CHEX 3268 and CHEX 5069), with raw counts (raw) shown in every facet as a common reference; facets are the three Tier families (%CD45, bead, vol). The dashed line marks a dissimilarity of 0.1 for reference. (F) Posterior contrasts of CD-CHEX lineage count stability for each scheme versus raw counts (model below). (G) Longitudinal stability of lineage and subpopulation counts from fresh blood of 10 donors (populations as in ). X-axis, visit in chronological order; y-axis, mean Bray–Curtis dissimilarity to the first visit across donors, with the shaded ribbon the standard error across donors; each line is one scheme (color), raw counts repeated in each facet as reference; facets are level (lineages, subpopulations) × Tier family. (H) Corresponding posterior contrasts versus raw counts. In (F, H) , Bray–Curtis dissimilarity to the reference timepoint was modelled by Beta regression (brms) with normalization scheme as a fixed effect and a random intercept per population group (CD-CHEX lot in F; donor in H). Points are the posterior median difference from raw counts (ΔBray–Curtis) and bars the 80% (thick) or 95% (thin) credible interval (CI); negative values indicate greater stability than raw. A scheme was classed as more stable (green) or less stable (orange) than raw when its 95% CI excluded zero, and equivalent (gray) when it included zero. P denotes the posterior probability that a scheme has lower temporal dissimilarity than raw (the proportion of posterior draws with ΔBray–Curtis < 0), such that P = 1.00 indicates every draw favored greater stability. All models converged well (Rˆ ≤ 1.01, no divergent transitions); model convergence and fit diagnostics are given in .

Article Snippet: CD-CHEX Plus stabilized human blood (213323, lots 32680071, 41550071, 50690071 and 53210071, Streck) was used as a biological-invariant control.

Techniques: Flow Cytometry, Stability, Counting

Journal: The Journal of Biological Chemistry

Article Title: The protease cathepsin K can debulk the cancer glycocalyx

doi: 10.1016/j.jbc.2026.111206

Figure Lengend Snippet: Only cathepsin K degrades cell-surface mucins on K562s. A, schematic describing the flow cytometry assay used to evaluate the degradation of cell-surface mucins on K562 cells by cathepsins. B, cell viability of cells following enzymatic treatment. Normalized staining for ( C ) MUC1, ( D ) CD43, and ( E ) total mucins via StcE E447D following enzymatic treatments for 3 h at pH 6 in Hanks' balanced salt solution (HBSS). Staining was normalized such that PBS-treated and fluorescence minus-one controls are defined as 100% and 0% staining within each replicate ( n = 3–4 biologically independent replicates). Cathepsins have been labeled with their letter, for example, “A” refers to cathepsin A. CTSE was excluded because of the low pH of the CTSE activation buffer causing cellular toxicity . See for representative flow cytometry histograms, assays performed at pH 5 and 7, and bar graphs with raw median fluorescence intensity (MFI) values. Data are shown as mean ± SD from three to four biologically independent replicates, and each dot represents a single replicate. p Values determined via one-way ANOVA corrected via Dunnett’s multiple comparison test, with a single pooled variance. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. CTSE, cathepsin E; StcE E447D , inactive point mutant of StcE used as a pan-mucin probe.

Article Snippet: Recombinant PSGL-1 (10 μg; Sino Biological; 13863-H08H), recombinant MUC1 (Sino Biological; 12123-H05H), recombinant CD43 (R&D Systems; 9680-CD-050), fetuin (Promega; V4961), and purified MUC16 (purified from OVCAR-3 as above) were mixed with CTSK or PBS control at a 1:50 CTSK:substrate molar ratio in 1:1 PBS:100 mM sodium acetate with a final pH of 5 (buffer pHed as above).

Techniques: Flow Cytometry, Staining, Fluorescence, Labeling, Activation Assay, Comparison, Mutagenesis