3150020b  (fluidigm)

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A and B) Violin plots reveal expression levels of five EMT/stemness and two polarization markers of macrophages in the four AXL-pSTAT3 categories in lung TMEs. Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (C) CD86 and CD163 correlation scatterplot of macrophages demonstrates the four subtypes based on mean values of CD86 and CD163 and bar graph of the subtype proportions in the four AXL-pSTAT3 categories. (D) Violin plots of expression levels of the seven markers in group 1 and 2 tumors. Data are mean ± SD; ***p < 0.001; Student’s t test. (E) Circle plots showing the proportions and expression levels of AXL, pSTAT3, and the seven markers of the four AXL-pSTAT3 categories in individual tumors from group 1 and 2 patients. (F and G) Diffusion maps of the pseudotime trajectories of the overall macrophages and macrophages from group 1 or 2 tumors of the four AXL-pSTAT3 categories. The arrows indicate the trajectory. (H) Boxplot of Shannon indices in the four AXL-pSTAT3 categories (n = 15 for each category). Data are mean ± SD; *p < 0.05; one-way ANOVA followed by Duncan’s multiple range tests. (I) Boxplot of Shannon indices in group 1 (n = 40) and 2 (n = 20) tumors. Data are mean ± SD; **p < 0.01; Student’s t test.

Article Snippet: Anti-Human CD86 150Ne , Fluidigm , Cat#3150020B; RRID: AB_2661798.

Techniques: Expressing, Diffusion-based Assay

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A) Co-cultured conditions of U937-derived macrophages and A549 lung cancer cells or MRC-5 lung fibroblasts with cytometry by time-of-flight (CyTOF) workflow. (B) Violin plots showing the expression levels of AXL and pSTAT3 of macrophages in condition 1, 3, and 5. Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (C) AXL and pSTAT3 correlation scatterplot of macrophages showing the four categories based on mean values of AXL and pSTAT3 and bar graph of category proportions in condition 1, 3, and 5. (D and E) Violin plots of expression levels of the seven AXL-pSTAT3-related markers in macrophages of condition 1, 3, and 5. Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (F) CD86 and CD163 correlation scatterplot of macrophages showing the four subtypes based on mean values of CD86 and CD163 and bar graph of subtype proportions in condition 1, 3, and 5. (G) Circle plots showing the proportions and expression levels of AXL, pSTAT3, and the seven markers of the four AXL-pSTAT3 categories in condition 1, 3, and 5. (H) Boxplot of Shannon indices in condition 1, 3, and 5 of 32 PhenoGraph clusters (n = 32). Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests.

Article Snippet: Anti-Human CD86 150Ne , Fluidigm , Cat#3150020B; RRID: AB_2661798.

Techniques: Cell Culture, Derivative Assay, Cytometry, Expressing

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A and B) IL-11 secretion was attenuated by dubermatinib (40 nmol/L) from A549 lung cancer cells (n = 2) and MRC-5 lung fibroblasts (n = 4). Data are mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test for each time point. (C and D) Kaplan-Meier curves depict overall and disease-free survival probability in TCGA lung adenocarcinoma cohort based on high ( Z score > 1) and low ( Z score < 1) IL11 expression of lung tumors. (E) Flow chart of induction of U937-derived macrophages and IL-11 treatment for CyTOF analysis. (F) AXL and STAT3 correlation scatterplot of U937-derived macrophages. (G) Violin plots showing the expression levels of AXL and STAT3 without and with IL-11 treatment (25 ng/mL). Data are mean ± SD; ***p < 0.001; Student’s t test. (H) Western blot analysis of IL-11-activated AXL-STAT3 signaling, i.e., phosphorylation of AXL and STAT3 (pSTAT3) in U937-derived macrophages. The cleavage product, phosphorylated AXL intracellular domain (pAXL-ICD), was observed. (I and J) Violin plots of expression levels of the seven AXL-STAT3-related markers without and with IL-11 treatment (25 ng/mL). Data are mean ± SD; ***p < 0.001; Student’s t test. (K) CD86 and CD163 correlation scatterplot of macrophages showing the four subtypes based on mean values of CD86 and CD163 and bar graph of subtype proportions of macrophages without and with IL-11 treatment (25 ng/mL).

Article Snippet: Anti-Human CD86 150Ne , Fluidigm , Cat#3150020B; RRID: AB_2661798.

Techniques: Expressing, Derivative Assay, Western Blot, Phospho-proteomics

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet:

Article Snippet: Anti-Human CD86 150Ne , Fluidigm , Cat#3150020B; RRID: AB_2661798.

Techniques: Recombinant, Reverse Transcription, Cell Viability Assay, In Situ, SYBR Green Assay, RNA Sequencing, Quantitative RT-PCR, Luciferase, Software

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet:

Article Snippet: Anti-human CD86 (clone IT2.2) (150ND) , Fluidigm , Cat# 3150020; RRID: AB_2687852.

Techniques: Recombinant, Purification, Staining, Cell Isolation, Transgenic Assay, Software, Gene Expression

Journal: STAR Protocols

Article Title: Mass cytometry staining for human bone marrow clinical samples

doi: 10.1016/j.xpro.2022.101163

Figure Lengend Snippet:

Article Snippet: Anti-Human CD138/Syndecan-1 (DL-101)-150Nd—25 tests (1:100) , Fluidigm , Cat#3150012C.

Techniques: Recombinant, Saline, Red Blood Cell Lysis, Staining, Blocking Assay, Antibody Labeling, Software, Cell Counting, Cytometry

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet: Pediatric AITD patients have altered frequencies of potentially pathogenic B cell subsets (immature, Bnd, and DN2) (A) Frequencies of immature (CD27 − IgM hi CD38 hi ), naive (CD27 - ), memory (CD27 + ), class-switched memory (CD27 + IgD − ), and plasmablast (CD27 hi CD38 hi ) B cells in adult-onset AITD (AA), adult healthy controls (AH), pediatric-onset AITD (PA), and pediatric healthy controls (PH) reveals PA patients have increased frequency of immature B cells compared to age matched controls. (B) Frequencies of anergic Bnd (CD27 − IgM lo IgD + ), DN2 (CD27 − IgD - CD21 − CXCR5 - ), and CD21- (CD19 + CD21 - ) B cells in AITD patients and controls reveals PA patients have reduced frequencies of Bnd and DN2 B cells compared to PH. (C) Mean metal intensity of CD86 and PTEN in CD19 + B cells and frequency of PD-1+ B cells is similar between AITD patients and controls, suggesting total B cells do not show increased activation in AITD patients. (D) B cells from AITD patients do not secrete increased levels of the pro-inflammatory cytokines, IFN-y, IL-6, or TNF-a, at baseline compared to controls. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1 . Frequency of B cell subsets, activation markers, and cytokine production were determined by manual gating, as depicted in and . See also Figure S7 for comparison of manually gated B ND cells to algorithm determined B ND cells. Statistical significance determined by One-way ANOVA with Sidak multiple comparisons post-test. ∗p < 0.05

Article Snippet: Mouse monoclonal anti-human CD86 (clone IT2.2) , Fluidigm , Cat#3150020B RRID: AB_2687852.

Techniques: Activation Assay, Comparison

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet: Unsupervised clustering and dimension reduction analysis of B cells reveals similar frequencies of B cell subpopulations in AITD patients and controls (A) B cells were manually gated as CD45 + CD19 + CD3-CD14-and then analyzed using the unsupervised clustering and dimension reduction programs PhenoGraph and X-shift to generate distinct clusters based on B cell surface marker expression (PD-1, IgM, CD27, CD21, CD38, CXCR5, CD11c, CD86, and IgD). The UMAP plot was created using total B cells from concatenated adult AITD, adult healthy control, pediatric AITD, and pediatric healthy control samples with a minimum of 5,000 B cell events. Eight unique B cell subpopulations were identified. UMAP plots of each surface marker are displayed to demonstrate varying expression levels among the B cell subpopulations. (B) Heatmap of B cell surface marker expression for identification of the 8 B cell subpopulations identified using PhenoGraph/X-Shift. DN: double-negative (CD27-IgD-CD21-), DN2: double-negative CD11c+ (CD27-IgD-CD21-CD11c+). (C) Frequencies of B cell subpopulations in adult AITD (AA), adult healthy control (AH), pediatric AITD (PA), and pediatric healthy control (PH) subjects demonstrate AA patients have increased mature naive B cells and decreased class-switched B cells compared to AH. Data are represented as mean ± SEM. Samples were processed as depicted in Figure S1 . See also Figure S7 for comparison of manually gated B ND cells to algorithm determined B ND cells. Statistical significance determined by Mann-Whitney non-parametric unpaired Student's t tests.

Article Snippet: Mouse monoclonal anti-human CD86 (clone IT2.2) , Fluidigm , Cat#3150020B RRID: AB_2687852.

Techniques: Marker, Expressing, Control, Comparison, MANN-WHITNEY

Journal: iScience

Article Title: Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD

doi: 10.1016/j.isci.2021.103626

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-human CD86 (clone IT2.2) , Fluidigm , Cat#3150020B RRID: AB_2687852.

Techniques: Recombinant, Antibody Labeling, Immunopeptidomics, Mass Cytometry, Software