Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: bioRxiv

Article Title: Transglutaminase 2 associated with PI3K and PTEN in a putative membrane-bound signalosome platform blunts cell death

doi: 10.1101/2022.02.16.480667

Figure Lengend Snippet: ( A ) Relative mRNA expression of CD11b at the indicated days by RT-qPCR normalized to GAPDH (n=3). ( B ) Flow cytometry analysis of cell surface expression of the differentiation marker CD11b/CD18 (n=3). ( C ) Relative mRNA expression of CD11c measured by RT-qPCR normalized to GAPDH (n=3). ( D ) Flow cytometry analysis of cell surface expression of differentiation marker CD11c/CD18 (n=3). Measurements were conducted in triplicate; values were validated by Flowing software 2.5.1. Statistical analysis was conducted by two-way ANOVA (Bonferroni post hoc test; *p <0.05, **p <0.01 and ***p <0.001, ****p <0.0001).

Article Snippet: F4/80 - cells were sorted and labelled with CD11c-PE and CD11b-FITC antibodies (1:25; R&D Systems) and APC-conjugated Annexin-V (Biolegend) for 15 min at 4°C in the dark, and then analysed with a BD FACSAria III flow cytometer.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Marker, Software

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet: Flow cytometry enriched antibody cocktail (22 μL) panel for analyzing rarer granulocyte and myeloid cells in mouse spleen for downstream scRNA-seq

Article Snippet: CD117 Antibody, anti-mouse, APC, REAfinityTM Clone REA791 , Miltenyi Biotec , Cat# 130-111-694; RRID: AB_2654592.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet:

Article Snippet: CD117 Antibody, anti-mouse, APC, REAfinityTM Clone REA791 , Miltenyi Biotec , Cat# 130-111-694; RRID: AB_2654592.

Techniques: Recombinant, Sterility, Saline, Extraction, Blocking Assay, Software, Solvent, Sequencing, RNA Sequencing, Transferring, Aerosol, Irradiation, Inverted Microscopy, Flow Cytometry, Fluorescence

Journal: Journal of Advanced Research

Article Title: Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

doi: 10.1016/j.jare.2024.08.023

Figure Lengend Snippet: Combined treatment overcomes TAM-related ibrutinib resistance in vivo. (A) The in vivo experimental design is presented. (B) The tumor growth curve of MCL tumors from NCG mice (n = 5) is shown. (C) The weights of xenograft MCL tumors from NCG mice (n = 5) are reported. (D) Representative images of tumors from NCG mice (n = 5) are displayed. (E) Flow cytometry was used to monitor macrophage polarization using peripheral blood specimens. (F) Flow cytometry was employed to analyze macrophage polarization in tumor tissue. (G) An ELISA assay was conducted to monitor the concentrations of CXCL5 and IL-8 in peripheral blood. (H) CXCL5 and CXCR2 expression in the tumor microenvironment was analyzed by immunohistochemistry (IHC). Scale bar = 100 μm. (I) The percentages of CXCL5 + TAMs and CXCR2 + TAMs in the tumor microenvironment were analyzed by flow cytometry. **** p < 0.0001.

Article Snippet: All mice were treated with ibrutinib (12.5 mg/kg, Selleck; Houston, TX, USA) daily via oral gavage.

Techniques: In Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

doi: 10.1016/j.jare.2024.08.023

Figure Lengend Snippet: Up-regulation of CXCL5 led to ibrutinib resistance. (A) Representative images of two independent cytokine array assays are shown. (B) The fold changes of CXCL5, CCL3, CXCL1, and CCL5 in the co-culture group compared to the control group were analyzed using the cytokine array. (C) ELISA assays measured cytokine levels at various time points. (D) Cell viability in MCL cells treated with ibrutinib was examined after selectively blocking the CXCL5, CCL5, CCL3, and CXCL1 in the co-culture system. (E) Western blot analysis confirmed CXCL5 expression in macrophages. (F) The in vivo experimental design is outlined. (G) The tumor growth curve of MCL tumors from NCG mice (n = 5) is presented. (H) The xenograft weights of MCL tumors from NCG mice (n = 5) are presented. (I) Representative images of tumors from NCG mice (n = 5) are displayed. (J) Flow cytometry was used to monitor macrophage polarization using peripheral blood specimens. (K) Flow cytometry was employed to analyze macrophage polarization in tumor tissues. (L) An ELISA assay monitored the concentrations of CXCL5 and IL-8 in peripheral blood. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: All mice were treated with ibrutinib (12.5 mg/kg, Selleck; Houston, TX, USA) daily via oral gavage.

Techniques: Co-Culture Assay, Control, Enzyme-linked Immunosorbent Assay, Blocking Assay, Western Blot, Expressing, In Vivo, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

doi: 10.1016/j.jare.2024.08.023

Figure Lengend Snippet: Co-culture of MCL cells with macrophages. (A-B) The M2-to-M1 ratio of macrophages from both ibrutinib-sensitive and −resistant subjects was analyzed using macrophage samples derived from lymphoma and blood. (C) Enrichment analysis of the KEGG signaling pathways conducted using the “clusterProfiler” package revealed significantly enriched KEGG pathways associated with correlated protein-coding genes. (D) A schematic diagram of the co-culture system illustrates MCL cells located on the upper side and macrophages on the lower side, separated by polycarbonate pore membranes with a pore size of 0.4 μm. (E) Mino and JEKO-1 cells were treated with the indicated concentrations of ibrutinib, with or without macrophages. Cell viability was evaluated using the CCK-8 assay, and control cells were treated with DMSO. (F-G) Cell apoptosis ratios were analyzed by flow cytometry. Mino and JEKO-1 cells were treated with either DMSO or 1 μM ibrutinib, and the percentages of Annexin V/PI (propidium iodide)-positive cells were measured. ****p < 0.0001.

Article Snippet: All mice were treated with ibrutinib (12.5 mg/kg, Selleck; Houston, TX, USA) daily via oral gavage.

Techniques: Co-Culture Assay, Derivative Assay, Protein-Protein interactions, Pore Size, CCK-8 Assay, Control, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

doi: 10.1016/j.jare.2024.08.023

Figure Lengend Snippet: Combination of CXCR2 inhibitor and ibrutinib displayed high synergistic inhibition of MCL growth. (A, B) The apoptosis of ibrutinib-treated cells was assessed using flow cytometry following treatment with CXCL5 or co-culture conditions. (C) Caspase-3 activity in Mino and JEKO-1 cells was measured under CXCL5 or co-culture treatments. (D) Compusyn software was employed to analyze the inhibition rates of malignant cells (MCLs) under ibrutinib, SB225002, and the combination of ibrutinib and SB225002 in a co-culture system. (E) The combined drug index (CI) of ibrutinib combined with SB225002 was analyzed using Compusyn software at different inhibition rates. (F) The dose-reduction index (DRI) of ibrutinib and SB225002 is presented. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Fa: Fraction affected.

Article Snippet: All mice were treated with ibrutinib (12.5 mg/kg, Selleck; Houston, TX, USA) daily via oral gavage.

Techniques: Inhibition, Flow Cytometry, Co-Culture Assay, Activity Assay, Software

Journal: Journal of Advanced Research

Article Title: Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

doi: 10.1016/j.jare.2024.08.023

Figure Lengend Snippet: CXCL5-induced ibrutinib resistance is independent of BTK pathways. (A) Gene Set Enrichment Analysis (GSEA) was performed using GSEA v3.0 software ( http://www.broad.mit.edu/gsea ). (B) The phosphorylation levels of BTK, p65, Akt, p38 MAPK, and STAT3 in Mino cells treated with CXCL5 (100 ng) and/or ibrutinib (1 μM) were analyzed by Western blotting. (C) The expression levels of p-Akt, p-p38 MAPK, and p-STAT3 after treatment with SB225002 (2.5 μM) and/or ibrutinib (1 μM) were analyzed by Western blotting. (D, E) KEGG analysis was conducted based on high-throughput sequencing assays using RNA extracted from cells treated with CXCL5 (100 ng) or SB225002 (2.5 μM). (F) The effects of ibrutinib and/or SB225002 on macrophage polarization were detected by flow cytometry in vitro. (G) The influence of CXCL5, ibrutinib, and SB225002 on CXCR2 expression was analyzed by Western blotting. ****p < 0.0001.

Article Snippet: All mice were treated with ibrutinib (12.5 mg/kg, Selleck; Houston, TX, USA) daily via oral gavage.

Techniques: Software, Phospho-proteomics, Western Blot, Expressing, Next-Generation Sequencing, Flow Cytometry, In Vitro

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet:

Article Snippet: Anti-human Vδ2 - FITC (clone 123R3) , Miltenyi Biotec , Cat#130-125-095; RRID: AB_2819745.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Multiplexing, Recombinant, Antibody Labeling, Staining, Amplification, Software, Flow Cytometry

Journal: BioMed Research International

Article Title: Cholesteryl Pullulan Encapsulated TNF- α Nanoparticles Are an Effective Mucosal Vaccine Adjuvant against Influenza Virus

doi: 10.1155/2015/471468

Figure Lengend Snippet: Flow cytometry of NALT cells after immunization with TNF/CHP nanoparticles. Mice were nasally administered with IVV (0.3 μ g/mouse) or IVV and TNF/CHP nanoparticles (5 μ g/mouse as TNF- α ) once a week for 3 times. Sixty-eight hours after the last immunization, NALT cells were prepared and subjected to flow cytometric analysis. The expression of CD11c and CD80/CD86 was examined. (a) and (d), saline; (b) and (e), IVV; (c) and (f), IVV with TNF/CHP nanoparticles; (a), (b), and (c), CD80 + /CD11c + ; and (d), (e), and (f), CD86 + /CD11c + .

Article Snippet: After being blocked with FcR blocker (Milteny Biotec, Bergisch Gladbach, Germany), the cells were stained with fluorescein isothiocyanate- (FITC-) labeled anti-mouse CD11c antibody and FITC-labeled anti-mouse CD80 antibody, or FITC-labeled anti-mouse CD86 antibody (Milteny Biotec), and subjected to flow cytometry (FCM) analysis using a flow cytometer (GALLIOS, Beckman Coulter, Brea, CA).

Techniques: Flow Cytometry, Expressing, Saline

Journal: BioMed Research International

Article Title: Cholesteryl Pullulan Encapsulated TNF- α Nanoparticles Are an Effective Mucosal Vaccine Adjuvant against Influenza Virus

doi: 10.1155/2015/471468

Figure Lengend Snippet: Antigen uptake of NALT and nasal passage DCs after TNF/CHP nanoparticles administration. Mice were immunized for 6 h with 10 μ g of Alexa-labeled OVA antigen for nasal administration with or without TNF/CHP nanoparticles as an adjuvant. The NALT and the nasal passage cells were prepared and subjected to FCM analysis with Gatelogic software and antigen uptake was measured by the Alexa 647 florescence intensity for DCs (CD11c + cells) detected in parallel, that is, ratio of Alexa 647 + /CD11c + . (a) NALT DCs, (b) nasal passage DCs. MFI, mean fluorescence intensity (mean ± SD, n = 4).

Article Snippet: After being blocked with FcR blocker (Milteny Biotec, Bergisch Gladbach, Germany), the cells were stained with fluorescein isothiocyanate- (FITC-) labeled anti-mouse CD11c antibody and FITC-labeled anti-mouse CD80 antibody, or FITC-labeled anti-mouse CD86 antibody (Milteny Biotec), and subjected to flow cytometry (FCM) analysis using a flow cytometer (GALLIOS, Beckman Coulter, Brea, CA).

Techniques: Labeling, Adjuvant, Software, Fluorescence

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: (A) CITE-seq analysis (protein) of hepatic Lin − CD45 + lymphocytes showing relative expression of CD49a, CD94, and NKp80 in the LT-ILC1 cluster illustrated in UMAP (only batch 2). (B) Relative expression levels of the indicated markers among each of the indicated populations as measured by CITE-seq analysis (only batch 2). (C) PhenoGraph cluster analysis of flow cytometry data from hepatic Lin − CD45 + lymphocytes displayed in UMAP. Clustering and UMAP is based on following markers: CD94, CD45, CD56, CD117, CD52, CD200R1, CRTH2, T-BET, NKp80, CD161, CD49a, EOMES, CXCR6, and CD127. (D) Heatmap showing the relative expression of the indicated markers from nine PhenoGraph clusters identified in (C) as measured by flow cytometry and calculated to a Z score for each marker. (E) Representative flow cytometry plots showing the gating strategy from singlet Lin − CD45 + lymphocytes (from healthy livers) to identify liver-type (LT)-ILC1 (pink), cNK (turquoise), CD49a + trNK cells (violet), CD49a − trNK cells (red), classical (c) ILC1 (gray), ILC2 (blue), and ILC3 (green) populations. (F) PhenoGraph cluster analysis of flow cytometry data from hepatic Lin − CD45 + lymphocytes with assignment of the indicated populations. (G) Representative histograms showing the relative expression of the indicated markers in each identified population to distinguish ILCs from NK cell populations. (H) Representative histograms showing the relative expression of the indicated markers in each identified population, each of which is elevated on LT-ILC1. See .

Article Snippet: NKp80 FITC (4A4.D10) , Miltenyi , RRID: AB_10829948; Cat#130-094-843.

Techniques: Expressing, Flow Cytometry, Marker

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: (A) Comparison of LT-ILC1 (Lin − CD45 + CD94 + NKp80 − CD200R1 + CD49a + ) frequencies calculated as percentages among total Lin − lymphocytes between control livers (perfusate n = 7), control livers (non-perfusate; n = 8), and cirrhotic livers (n = 29) (patient characteristics shown in ). (B) Correlation matrix estimated for LT-ILC1s (percent of Lin − ), age, AST, ALT, gamma-GT, total bilirubin, MELD score (Model of End Stage Liver Disease), IFNG (mRNA), IL13 (mRNA), IL1B (mRNA), IL2 (mRNA), IL7 (mRNA), and TGFB1 (mRNA). LT-ILC1 frequencies were obtained from 25 cirrhotic livers. Spearman correlation coefficients (R) are given as color code. Statistically significant correlations are indicated with *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative expression of the indicated markers by human LT-ILC1 (pink), cNK cells (turquoise), CD49a + trNK cells (violet), CD49a − trNK cells (red), cILC1s (gray), ILC2s (blue), and ILC3s (green), comparing populations derived from healthy and cirrhotic livers. Data are representative of at least three independent experiments. (D) Comparison of IFN-γ production (healthy, n ≥ 7; cirrhotic, n ≥ 9), IL-2 production (healthy, n = 4; cirrhotic, n ≥ 4), CD107a degranulation (healthy, n = 3; cirrhotic, n = 5), TNF-α production (healthy, n ≥ 7; cirrhotic, n ≥ 5), and GM-CSF production (healthy, n ≥ 4; cirrhotic, n ≥ 3) between healthy and cirrhotic livers related to LT-ILC1 (pink), cNK cells (turquoise), CD49a + trNK cells (violet), CD49a − trNK cells (red), and ILC3s (green), from healthy and cirrhotic livers following 5 h PMA and ionomycin stimulation and measured by intracellular flow cytometry. (E) Volcano plot showing DEG differences based on scRNA-seq data between LT-ILC1s comparing “control versus cirrhotic liver tissues” from batch 2. *p < 0.05, ***p < 0.001, error bars represent SEM. See .

Article Snippet: NKp80 FITC (4A4.D10) , Miltenyi , RRID: AB_10829948; Cat#130-094-843.

Techniques: Comparison, Control, Expressing, Derivative Assay, Flow Cytometry

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: (A) Sorted peripheral blood ILCPs ( ; Lin − CD45 + CD127 + CD94 − NKp80 − CD45RA + CD117 + CD294 − KLRG1 − NKP44 − ) from four healthy donors were cultured for 2 weeks on OP9-DL4 feeder cells or LSECs with IL-7 +/− TGF-β1 as shown to the left. The dot plots to the right show the expression patterns of CD56 and CD94 among Lin − lymphocytes derived in the indicated culture conditions. (B) Frequencies of CD200R1 + CD49a + EOMES − cells among total Lin − CD45 + CD94 + CD56 + cells derived in vitro following culture of ILCPs (n = 8) as in (A). (C) Representative flow cytometry analyses of EOMES, T-BET, perforin, CXCR6, NKp80, CD200R1, and CD49a expression by Lin − CD45 + CD94 + CD56 + cells derived from ILCPs in the indicated culture conditions. Fluorescence minus one (FMO) control with the respective lacking specific antibody is shown as black dotted lines in each histogram. (D) Intracellular flow cytometry analysis of IFN-γ, IL-2, GM-CSF, TNF-α, and IL-22 production by in vitro derived Lin − CD45 + CD94 + CD56 + cells generated from ILCPs as in (A) and following stimulation for 5 h with PMA and ionomycin. Unstimulated cells were set as controls (dotted black line). (E) Representative flow cytometry analyses showing expression of the indicated markers by total Lin − CD45 + cells obtained after 2 weeks culture of primary sorted liver-type hepatic cNK, CD49a + trNK, or CD49a − trNK cells on OP9-DL4 feeder cells or LSECs with IL-7 +/− TGF-β1 as shown to the left. Data are representative of at least three independent experiments. (F) Representative flow cytometry analyses showing expression of the indicated markers by total Lin − CD45 + cells obtained after 2-week culture of primary sorted liver-type ILC1s (LT-ILC1s) or hepatic NK cells (sorted as Lin − CD56 + CD94 + NKp80 + ) with OP9-DL4 cells and IL-7. Data are representative of at least three independent experiments. (G) Frequencies of CD200R1 + CD49a + EOMES − cells among total Lin − CD45 + CD94 + lymphocytes obtained from cultures initiated with either total hepatic NK cells or liver-type ILC1s (F). *p < 0.05, **p < 0.01, ***p < 0.001, error bars represent SEM. See .

Article Snippet: NKp80 FITC (4A4.D10) , Miltenyi , RRID: AB_10829948; Cat#130-094-843.

Techniques: Cell Culture, Expressing, Derivative Assay, In Vitro, Flow Cytometry, Fluorescence, Control, Generated

Journal: Cell reports

Article Title: Single-cell RNA sequencing identifies a population of human liver-type ILC1s

doi: 10.1016/j.celrep.2022.111937

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NKp80 FITC (4A4.D10) , Miltenyi , RRID: AB_10829948; Cat#130-094-843.

Techniques: Blocking Assay, Recombinant, Clinical Proteomics, Nucleic Acid Electrophoresis, Software, Inverted Microscopy

Journal: eLife

Article Title: HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties

doi: 10.7554/eLife.33947

Figure Lengend Snippet:

Article Snippet: For flow cytometry, mouse anti-human EPCAM (CD326) clone HEA-125 was purchased from Miltenyi Biotec (San Diego, CA).

Techniques: Clone Assay, Variant Assay, shRNA, Sequencing, Amplification, Transfection, Construct, Flow Cytometry, Western Blot, Mutagenesis, Recombinant, Plasmid Preparation, Blocking Assay, Control, Chromatin Immunoprecipitation, Magnetic Beads, Luciferase, SYBR Green Assay, Binding Assay, Software

Journal: Cell Host & Microbe

Article Title: Unveiling P. vivax invasion pathways in Duffy-negative individuals

doi: 10.1016/j.chom.2023.11.007

Figure Lengend Snippet:

Article Snippet: APC-conjugated recombinant human monoclonal anti-DARC antibody (flow cytometry) , Miltenyi Biotec , Cat.# 130-120-239 (clone REA376).

Techniques: Flow Cytometry, Recombinant, Western Blot, Fluorescence, Software, Microscopy, Imaging