Journal: Plant physiology

Article Title: EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

doi: 10.1093/plphys/kiac214

Figure Lengend Snippet: Figure 8 XLG2 functions in cerk1-4 cell death signaling together with PUB2. A, Venus-XLG2 and Venus-XLG2E293K were isolated from stably trans- formed Arabidopsis lines with anti-GFP nanobody beads. The samples were analyzed by LC–MS/MS and PSMs corresponding to XLG2/XLG2E293K, PUB4 and PUB2 were counted. Data are presented as average ±SD of three independent transgenic lines. Untransformed xlg2-2 served as a negative control. B, Western blot of the indicated lines with a specific XLG2 antibody. The experiment was done three times. C, pub2-1 and pub4-3 were crossed with cerk1-4. Single and double mutants as well as the background accessions Col-0 and Col-3 gl1 were inoculated with Blumeria graminis f. sp. hordei (Bgh) and analyzed at 7 dpi. Images of representative plants are shown. Inoculation experiments were performed twice with Bgh and twice with E. cruciferarum. D, 7 dpi Bgh infection, six whole rosettes per genotype were pooled and used for Western blots with a PR1-specific anti- body. CBB, Coomassie brilliant blue-stained membrane. PR1 accumulation was tested twice with Bgh and twice with E. cruciferarum.

Article Snippet: For GFP pull-downs, standard protein extracts were prepared (Petutschnig et al., 2010) and the target protein was isolated with either Chromotek GFP trap beads (mass spectrometry experiments) or Nanotag GFP selector beads (Co-IP experiments).

Techniques: Isolation, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Transgenic Assay, Negative Control, Western Blot, Infection, Staining, Membrane

Journal: Cell Reports Methods

Article Title: Application of spectral flow cytometry for comprehensive detection of immune metabolism in patient-derived microsamples

doi: 10.1016/j.crmeth.2026.101330

Figure Lengend Snippet: Assessing spectral resolvability of metabolic probes for multiparametric cytometry (A) Experimental workflow for evaluating probe co-detection. (B and D) Representative density plots demonstrating spectral overlap for pairwise probe combinations within the (B) FITC and (D) PE channel. (C and E) Complementary validation matrices showing computational spectral similarity scores from FluoroFinder (lower left quadrant) versus empirical resolvability determined by unmixing (upper right quadrant). Gray indicates non-resolvable pairs; colored tiles indicate resolvable pairs (C, green; E, red).

Article Snippet: Cells were washed and then acquired by full spectrum flow cytometry (SONY ID7000).

Techniques: Cytometry, Biomarker Discovery

Journal: Cell Reports Methods

Article Title: Application of spectral flow cytometry for comprehensive detection of immune metabolism in patient-derived microsamples

doi: 10.1016/j.crmeth.2026.101330

Figure Lengend Snippet: Validation framework for spectral co-resolvability of metabolic probes and fluorophore conjugates (A) Experimental workflow for co-detection assessment. (B, D, and F) Representative flow cytometry density plots demonstrating pairwise mixing between metabolic probes and fluorophores in the (B) FITC/AF488, (D) PE, and (F) APC/AF647 channel. (C, E, and G) Validation matrices per channel: upper, computational spectral similarity by FluoroFinder; lower, empirically determined resolvability. Gray tiles indicate non-resolvable pairs; colored tiles confirm resolvable pairs. (C) FITC/AF488, green; (E) PE, red; and (G) APC/AF647, blue.

Article Snippet: Cells were washed and then acquired by full spectrum flow cytometry (SONY ID7000).

Techniques: Biomarker Discovery, Flow Cytometry

Journal: Cell Reports Methods

Article Title: Application of spectral flow cytometry for comprehensive detection of immune metabolism in patient-derived microsamples

doi: 10.1016/j.crmeth.2026.101330

Figure Lengend Snippet: Validation of three metabolic probes for simultaneous assessment of mitochondrial activity and oxidative stress (A) Representative flow cytometry density plots demonstrating spectral resolution of co-stained probes. (B) Validation matrix: lower left quadrant, computational spectral similarity by FluoroFinder; upper right quadrant, empirical resolvability determination (red: resolvable pairs). (C) Mean fluorescence intensity (MFI; mean ± SD) of individual probes in control versus rotenone/antimycin A (Rot/AA)-treated groups ( n = 6). (D) Correlation analysis of MFI between single-stain and multiplexed conditions across probes (Spearman’s r). (E) MFI of probes (mean ± SD) in control versus EZH2-knockdown (EZH2-sh) groups under single and multiplexed staining. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: Cells were washed and then acquired by full spectrum flow cytometry (SONY ID7000).

Techniques: Biomarker Discovery, Activity Assay, Flow Cytometry, Staining, Fluorescence, Control, Knockdown

Journal: Indian Journal of Ophthalmology

Article Title: A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells

doi: 10.4103/ijo.IJO_2348_21

Figure Lengend Snippet: Evidence of Rb spontaneous metastasis in different parts of chick embryo analyzed using in vivo IVIS spectral imaging. Representative image of in vivo fluorescence observed in chick embryo transplanted with (a) CM-Dil Y79 CD133 - cells, (b) CM-Dil Y79 CD133 + cells, (d) eGFP Y79 CD133 - cells, and (e) eGFP Y79 CD133 + cells. Quantification of fluorescence intensity within the chick embryo revealed that CD133 - cells had increased spontaneous metastasis when compared to CD133 + cells (**P = 0.0077, 0.0017)

Article Snippet: The whole embryos were analyzed using the IVIS Spectrum in vivo optical imaging system (PerkinElmer) to track the presence of labeled tumor cells.

Techniques: In Vivo, Imaging, Fluorescence

Journal: Immunity

Article Title: Proteomic Analyses of Human Regulatory T Cells Reveal Adaptations in Signaling Pathways that Protect Cellular Identity.

doi: 10.1016/j.immuni.2018.04.008

Figure Lengend Snippet: Figure 1. Proteomic Profiles of CD4+ T Cell Subsets (A) Representative flow cytometric CD45RA and CD25 profiles of the human CD4+ T cell subsets subjected to MS analysis: nTconv (1, blue), mTconv (2, cyan), Fr. III population (3, green), nTreg (4, red), and eTreg (5, orange) cells. (B) PCA plot of proteomes (each square represents subset of a single donor). (C) Heatmap depicting PC coefficients of subset proteomes. (D) Hierarchical clustering and heatmap showing relative protein expression values (z-score and log2-transformed LFQ protein intensities) of the 422 differentially expressed proteins (FDR < 0.05; S = 0) between the subsets. Columns correspond to samples from individual donors (D1–3). Grey-scale boxes (left) indicate clusters. Dashed lines demarcate the common Treg cell protein signature (see Table S1). Data are from three independent experiments, each with technical triplicate samples.

Article Snippet: Sample preparation for mass spectrometry (MS) FACS-purified CD4+ subsets (at least 1x106 cells per subset) from 3-6 donors were placed in Protein LoBind tubes (Eppendorf), washed in PBS and lysed in 100mMTris HCl pH 8.0 with 4%SDS, 100mMDTT.

Techniques: Expressing, Transformation Assay

Journal: Immunity

Article Title: Proteomic Analyses of Human Regulatory T Cells Reveal Adaptations in Signaling Pathways that Protect Cellular Identity.

doi: 10.1016/j.immuni.2018.04.008

Figure Lengend Snippet: Figure 4. The Common Treg Cell Protein Signature (A) Unsupervised hierarchical clustering and heatmap of expression levels (z-scores) of the 51 proteins in the common signature of nTreg and eTreg cells. (B) Functional and spatial representation of the common Treg cell signature proteins (red denotes higher and blue lower expression in Treg cells than the mean value in all five CD4+ T cell subsets). Proteins in gray refer to pathways in which signature proteins are known to operate. (C and D) Proteomes were measured from in vitro expanded Tconv and Treg cells, unstimulated (US) or stimulated for 24 hr with anti-CD3 and anti-CD28 mAbs. (C) Hierarchical clustering and heatmap showing the z-score and log2-transformed LFQ protein intensities of the 320 differentially expressed proteins (FDR < 0.05) between the indicated populations. GO terms corresponding to numbered clusters demarcated by dashed lines are shown in Figure S4H. Data are from three independent experiments, each with biological triplicates. (D) Unsupervised hierarchical clustering and heatmap of relative expression levels (z-scores) in expanded cells of 46 proteins detected from the common Treg cell signature. Asterisks indicate significantly different expression between in vitro expanded Treg and Tconv cells (FDR < 0.05). Proteins in red deviate from the expression pattern in the ex vivo common Treg cell signature.

Article Snippet: Sample preparation for mass spectrometry (MS) FACS-purified CD4+ subsets (at least 1x106 cells per subset) from 3-6 donors were placed in Protein LoBind tubes (Eppendorf), washed in PBS and lysed in 100mMTris HCl pH 8.0 with 4%SDS, 100mMDTT.

Techniques: Expressing, Functional Assay, In Vitro, Transformation Assay, Ex Vivo

Journal: Immunity

Article Title: Proteomic Analyses of Human Regulatory T Cells Reveal Adaptations in Signaling Pathways that Protect Cellular Identity.

doi: 10.1016/j.immuni.2018.04.008

Figure Lengend Snippet: Figure 5. The eTreg Cell Protein Signature (A) Heatmap showing z-score and log2-transformed LFQ protein intensities across the five CD4+ T cell populations of preferentially high (eTreghi) or low (eTreglo) abundance proteins in eTreg cells (center three columns). Each column corresponds to cells from a different donor (D1–3). (B) GO analysis (STRING) of pathways (confidence view) enriched within eTreghi (top) and eTreglo (bottom) protein clusters. Thicker lines represent stronger associations. (C) Estimated copy number ratio of FOXP3-interacting transcription factors to FOXP3 in nTreg (red), eTreg (orange), and mTconv (cyan) cells, determined by the proteomic ruler approach (Wisniewski et al., 2014). Dashed line represents a ratio of 1. Each symbol in represents an individual donor (same as Figure 1).

Article Snippet: Sample preparation for mass spectrometry (MS) FACS-purified CD4+ subsets (at least 1x106 cells per subset) from 3-6 donors were placed in Protein LoBind tubes (Eppendorf), washed in PBS and lysed in 100mMTris HCl pH 8.0 with 4%SDS, 100mMDTT.

Techniques: Transformation Assay

Journal: Immunity

Article Title: Proteomic Analyses of Human Regulatory T Cells Reveal Adaptations in Signaling Pathways that Protect Cellular Identity.

doi: 10.1016/j.immuni.2018.04.008

Figure Lengend Snippet: Figure 7. Proteomics Reveals Functional Heterogeneity among Fr.III and eTreg Cells (A) Hierarchical clustering and heatmap showing z-score and log2-transformed LFQ protein intensities of 164 differentially expressed proteins (FDR < 0.05; S = 0.4) among 6 CD4+ T cell subsets defined as indicated. Each column corresponds to cells from a different donor (n = 3–6). Arrow highlights a cluster differentiating eTreg cell and Fr.III CD127 cells. (B) PCA plot of the six CD4+ T cell subset proteomes (squares represent different donors). (C) Heatmap representing percentage of FOXP3 TSDR DNA methylation per subset (see color scale). (D) Heatmap of average CCR4 and CD49d expression levels (z-scores) measured by MS. (E) Representative FACS plots of CD49d and CCR4 in CD127+ Fr.III (dark green), CD127 Fr.III (light green), and CD127 eTreg cells (orange). (F) Representative FACS plots of cytokines produced by the different cell subsets stimulated with PMA and Ionomycin for 4 hr (total) or gated for CD49d and CCR4. (G) Percentage of cells IFN-g-, IL-17-, and IL-2-positive cells after 4 hr of treatment with PMA and ionomycin (mean ± SEM of n = 3 experiments). Statistical analysis by two-way ANOVA followed by a Turkey’s multiple comparison test (**p < 0.01; ***p < 0.001; indicate significant differences compared to the total population; #p < 0.05 indicate significant differences compared to CCR4+CD49d population).

Article Snippet: Sample preparation for mass spectrometry (MS) FACS-purified CD4+ subsets (at least 1x106 cells per subset) from 3-6 donors were placed in Protein LoBind tubes (Eppendorf), washed in PBS and lysed in 100mMTris HCl pH 8.0 with 4%SDS, 100mMDTT.

Techniques: Functional Assay, Transformation Assay, DNA Methylation Assay, Expressing, Produced, Comparison