Fig 2: Overview of 19-parameter gating strategy developed for the characterization of T-cell, B-cell, and ASC subsets within freshly isolated splenocytes from 21-day-old BALB/c mice. 1 × 106 splenocytes were incubated in Fc Block™, followed by fixable viability stain (FVS) and a 17-parameter extracellular antibody cocktail containing 10% brilliant stain buffer plus (BD biosciences). Intracellular staining was performed following fixation-permeabilization of extracellular stained splenocytes. Data were acquired on a BD LSRFortessa™ (BD Biosciences). (A–C) Removal of cellular debris, doublets, nonviable cells and stromal cells. (D) Primary delineation of TCRß-CD19+, TCRß+CD4+, and TCRß-CD4/CD19- cells. (E–M) Characterization of (E) plasma cells, (F) plasmablasts, (G) activated B-cells, (H) memory B-cells, (I) naïve, effector memory and central memory T-cells, (J) effector and regulatory T-cells, (K) ICOS+CD44+ Treg, (L) T follicular helper cells, and (M) follicular regulatory T-cells. All plots are representative of individual samples. Manual gating was determined using fluorescence minus one (FMO) controls where necessary (Figure S4) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: Characterization of morphology, phenotype, and differentiation potential of UC-MSCs cultured in various xeno-free media. a A time schedule of the study. b UC-MSC morphology in xeno-free (M1, M2, M3, M4, M5) and control media (M6 and M7) at passage 1. Representative images in Nomarski Intereference Contrast are shown with a scale bar of 100 µm. c Surface antigen expression on UC-MSCs by flow cytometry. Cells were stained with selected antibodies conjugated with fluorochromes, acquired on BD LSRFortessa (BD Biosciences) and analyzed using the FlowJo software. Results are shown as a mean ± SD for cells cultured in control medium M6. Representative histograms are presented. d Differentiation potential of UC-MSCs into mesodermal lineages. Representative images of UC-MSCs differentiated into osteocytes, adipocytes and chondrocytes are shown as indicated (left). Scale bar shows 100 µm. Calcium phosphate deposits were stained with Alizarin Red-S, proteoglycans with Alcian Blue, and fat droplets were visible in bright field. Mean percentage of differentiation of five UC-MSCs lineages in all examined conditions (right). UC-MSCs umbilical cord-derived mesenchymal stem cells

Fig 4: Comparison of relationship between staining efficacy and parasite burden in samples acquired using BD LSRFortessa, BD LSRFortessa X-20 or BD FACSCanto II flow cytometer.Samples acquired with BD LSRFortessa (A), BD LSRFortessa X-20 (B) or BD FACSCanto II (C) flow cytometer were divided by percentage of Alexa 488+ events among events in Oocyst gate (staining efficacy). Parasite burden of samples categorized previously by staining efficacy and acquired with BD LSRFortessa (D), BD LSRFortessa X-20 (E) or BD FACSCanto II (F) flow cytometer. Staining efficacy (A to C) is inversely proportional to parasite burden (D to F) in same samples. ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001).

Fig 5: Gating schema for manual analysis of CyTOF data from tumour and blood specimens.A) Tumours were mechanically dissociated and cells were stained with immune cell-specific antibodies. Specimens were assayed on the Helios mass cytometer via CyTOF Software. Cytometer data were then prepared for manual and unsupervised analyses via FlowJo software. Major cell populations were identified manually and reported. An example of one patient specimen is shown above for reference. B) Gating schema for flow cytometric analysis of blood specimens. Peripheral blood mononuclear cells from patient specimens were stained along with FMO (fluorescence minus one) controls and assayed via a BD LSRFortessa cytometer and BD FACSDiva acquisition software. Data were analysed via FlowJo software as described above. Briefly, live CD3+ singlets were identified and gated into T cell lineages, and those lineages analysed for frequency of each of eight phenotypic markers (BCL6, BLIMP1, CD27, CD28, cMYC, EOMES, ICOS, Ki67) as defined by FMO (fluorescence minus one) specimens. An example of one phenotypic marker (EOMES) in one patient specimen is shown above for reference.