Journal: Journal of Nanobiotechnology

Article Title: Cu-doped TiO 2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies

doi: 10.1186/s12951-023-01844-z

Figure Lengend Snippet: A , B Histograms displaying the relative level of A) surface-located MHC-I, B) surface-located calreticulin in KLN 205 cells exposed to saline control, 33% Cu-doped TiO 2 NPs or 1 µM mitoxanthrone as positive control expressed as the relative level of cells positive for the marker out of the total population of cells analysed by ImageStreamX Mark II analysis. C , D Histograms displaying the relative level of C) autophagy marker LC3, D) autophagic flux marker p62 in KLN 205 cells exposed to saline control, 33% Cu-doped TiO 2 NPs or 10 nM rapamycin as positive control expressed as the relative level of cells positive for the marker out of the total population of cells analysed by ImageStreamX Mark II analysis. E Histogram showing the viability of KLN 205 cells treated with 33% Cu-doped TiO 2 NPs at the concentrations indicated in the presence and absence of caspase-inhibitor Z-VAD-fmk. Data are expressed as mean ± SEM ( n = 4). F Volcano plot for 84 genes related to cellular stress and toxicity analyzed in untreated KLN 205 cells and 33% Cu-doped TiO 2 NPs at 20 µg/ml for 24 h, where significant differences in expressed genes are indicated in orange (overexpression) or blue (underexpression). G The significant genes found in F) are further indicated with their respective fold difference compared to untreated KLN 205 cells. Data are expressed as mean ± SEM ( n = 4). H , I Histograms of RAW 264.7 macrophages either untreated or exposed to 33% Cu-doped TiO2 NPs at 40 µg/ml for 24 h, displaying the level of surface-located H) M2 macrophage markers (CD163, CD206) or I) M1 macrophage markers (CD86, MHC-II). Significant differences between a treated group and untreated controls at the same time point are indicated where relevant (p < 0.001: ***; p < 0.0001: ****) based on ANOVA testing using GraphPad Prism 9 ( n = 8)

Article Snippet: Media were removed, cells were washed with PBS, trypsinized and all supernatants, washing media and cells were added together and centrifuged at 2500 rpm for 6 min after which cells were stained with anti-CD86 antibody (FITC-anti-CD86 clone GL1, Thermo Fisher Scientific), anti-MHC II antibody (PE-Cy7-anti-MHC-II clone M5-114–15-2, Thermo Fisher Scientific) and anti-CD80 antibody (APC-anti-CD80 clone 16-10A1, Thermo Fisher Scientific) for 30 min at 4 °C.

Techniques: Positive Control, Marker, Over Expression

Journal: Journal of Nanobiotechnology

Article Title: Cu-doped TiO 2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies

doi: 10.1186/s12951-023-01844-z

Figure Lengend Snippet: A – C Histograms displaying the relative level of A) MHC-II, B) CD86, C) CD80 in in vitro cultured DCs exposed to saline (control) or 33% Cu-doped TiO 2 NPs. D Western blot of in vitro cultured DCs exposed to saline (control) or 33% Cu-doped TiO 2 NPs and analyzed for the expression level of NLRP3 and GAPDH as a housekeeping control. E Histogram showing the amount of IL1β released by in vitro cultured DCs either exposed to saline (control), 33% Cu-doped TiO 2 NPs or a positive control of LPS + ATP. F Histogram displaying the viability of in vitro cultured DCs exposed to 33% Cu-doped TiO 2 expressed relative to the viability of untreated DCs. G , H Histogram showing G) the amount of IL1β released or H) MHC-II levels by in vitro cultured DCs either exposed to saline (control) or 33% Cu-doped TiO 2 NPs in the presence of NLRP3-inhibitor MCC950. I , J Histograms indicating the level of I) IL12 and J) IL23 secreted by in vitro cultured DCs exposed to saline (vehicle control), 33% Cu-doped TiO 2 NPs or positive controls (100 ng/ml IL1β for I) and 300 µM wortmanin for J)). K–N Histograms indicating intracellular cytokine levels indicative of Th17 cell types obtained from tumor samples either treated with vehicle control (saline) or 33% Cu-doped TiO 2 NPs for K) IL17A, L) IL17F, M) IL21, N) IL22. These data are expressed as the level of cytokine-positive lymphocytes relative to the total number of lymphocytes. Significant differences between a treated group and untreated controls at the same time point are indicated where relevant (p < 0.05: *, p < 0.01: **; p < 0.001: ***; p < 0.0001: ****) based on ANOVA testing using GraphPad Prism 9 ( n = 6)

Article Snippet: Media were removed, cells were washed with PBS, trypsinized and all supernatants, washing media and cells were added together and centrifuged at 2500 rpm for 6 min after which cells were stained with anti-CD86 antibody (FITC-anti-CD86 clone GL1, Thermo Fisher Scientific), anti-MHC II antibody (PE-Cy7-anti-MHC-II clone M5-114–15-2, Thermo Fisher Scientific) and anti-CD80 antibody (APC-anti-CD80 clone 16-10A1, Thermo Fisher Scientific) for 30 min at 4 °C.

Techniques: In Vitro, Cell Culture, Western Blot, Expressing, Positive Control

Journal: Annals of Neurology

Article Title: B ‐cell very late antigen‐4 deficiency reduces leukocyte recruitment and susceptibility to central nervous system autoimmunity

doi: 10.1002/ana.24387

Figure Lengend Snippet: Selective α4‐deficiency on B cells reduces disease severity in recombinant human myelin oligodendrocyte glycoprotein (rhMOG)‐induced experimental autoimmune encephalomyelitis (EAE), whereas peripheral B‐cell properties appear to be unchanged. (A) α4 (CD49d) surface expression on CD19 + B220 + B cells and CD4 + T cells in the peripheral blood of naive CD19cre/α4 f/f or control mice assessed by flow cytometry. An isotype control antibody (ab) was used to define the negative population, and a healthy wild‐type (WT) mouse served as a positive control. Histograms show 1 representative mouse per group (n = 4 mice/group). (B) CD19cre/α4 f/f (n = 9 mice) and CD19cre/α4 f/WT control mice (n = 7 mice) were immunized with 100µg rhMOG (upper panel). CD19cre/α4 f/f (n = 17 mice) and CD19cre control mice (n = 10 mice) were immunized similarly (lower panel). In the lower panel, cumulative data from 2 independent experiments are shown. Similar results were obtained in another independent experiment with an α4 f/f control group (not shown). Data shown represent mean disease score ± standard error of the mean (SEM). ** p ≤ 0.01; Mann–Whitney U test; differences were significant ( p ≤ 0.05) for days 14 to 35, except day 28, in the upper panel and for days 12 to 16 in the lower panel. (C) Surface activation markers CD80 (B7‐1) and MHC II were assessed on B cells (gated on viable CD19 + B220 + cells) by flow cytometry in the spleen at peak of disease (day 14 postimmunization with 100µg rhMOG) in CD19cre/α4 f/f or control mice. Similar results were obtained in the blood, at a later time point (31 days postimmunization), and for CD86 (B7‐2; not shown). Flow cytometry plots show 1 representative mouse per group. Bar graphs represent mean ± SEM of n = 3 mice/group. ns = not significant; t test. (D) Th1 (IFN‐γ) and Th17 (IL‐17A) T‐cell differentiation is measured by intracellular cytokine staining in the spleen 31 days postimmunization. Flow cytometry plots show 1 representative mouse per group (gated on viable CD4 + T cells). Bar graphs represent mean ± SEM of n = 3 mice/group. ns = not significant; t test. Similar results were obtained at peak of disease (14 days postimmunization; not shown). (E) Serum immunoglobulin G (IgG) antibodies against rhMOG were detected by enzyme‐linked immunosorbent assay. The optical density (OD) at 450nm is indicated. Serum was obtained on day 31 postimmunization with 100µg rhMOG in CD19cre/α4 f/f or control mice (n = 3 mice/group) and diluted 1:9,000 before analysis. ns = not significant; t test. Control mice were CD19cre in all experiments, except in B, upper panel, where they were CD19cre/α4 f/WT . FS, forward scatter.

Article Snippet: Antibodies to mouse CD19 PerCP‐Cy5.5 (eBio1D3), B220 (CD45R) APC‐Cy7 (RA3–6B2), MHC‐II (I‐A/I‐E) PE‐Cy7 (M5/114.15.2), CD80 (B7‐1) APC (16‐10A1), CD4 APC‐Cy7 (RM4–5), and CD11b PE‐Cy7 (M1/70) were purchased from eBioscience (San Diego, CA).

Techniques: Recombinant, Expressing, Flow Cytometry, Positive Control, MANN-WHITNEY, Activation Assay, Cell Differentiation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage

doi: 10.1038/s41590-018-0276-y

Figure Lengend Snippet: a, Heatmap of SingleR annotation scores derived by reference to the ImmGen database with clusters superimposed on the t-SNE plot. MF = macrophages. b, Heatmap of genes differentially expressed between C1 and C3 with examples superimposed on the t-SNE plot. Cells are ordered by the number of non-zero C3 genes. c, Percentage of cells in each cluster that express genes in b (differentially expressed between C1 and C3). Asterisk indicates that 45% of C2 cells express at least 33% of C1 genes and at least 33% of C3 genes. d, Heatmap of the expression of C3 genes in bulk RNA-seq of alveolar macrophages (SiglecF + CD11c + ) sorted on MHCII lo at baseline and both MHCII lo and MHCII hi at two time points after bleomycin lung injury (n=2 mice biologically independent mice for baseline, n=3 biologically independent mice at 2 weeks, and n=3 biologically independent mice at 4 weeks).

Article Snippet: Cells were passed through a 70 μm strainer and stained at 4 °C for 30 minutes with following antibodies (1:100): SiglecF-APC (catalog number 50–1702-82, clone 1RNM44N, eBioscience), MHCII-APC-Cy7 (catalog number 47–5321-82, clone M5/114.15.2, eBioscience), and CD11c-PE (catalog number 557401, clone HL3, BD Biosciences).

Techniques: Derivative Assay, Expressing, RNA Sequencing

Journal: Nature immunology

Article Title: Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage

doi: 10.1038/s41590-018-0276-y

Figure Lengend Snippet: a, Heatmap of C3 genes from bleomycin-induced fibrosis measured by microarray in an epithelial telomere dysfunction model of progressive lung fibrosis (n=3 biologically independent mice in each condition, center value is mean). b, Gene set enrichment analysis scores of human orthologues of cluster C1 genes and of MHCII genes in bulk RNA-seq samples from patient lung biopsy specimens . Points indicate individual microarray samples from n=167 and n=50 patients and controls, respectively. c, Immunofluorescence of human fibrotic and healthy control lung (representative images are shown; quantitation is for n=3 independent fibrotic patient lung and n=3 independent healthy lung samples, 10 images per sample). Scale bar, 50 μm. SH=second harmonic imaging of collagen. Box plot center line is median, box limits are upper and lower quartiles, and whiskers denote the largest and smallest values no more than 1.5 times the interquartile range from the limits. Wilcoxon test 2-tailed p-values are presented. * p<0.01, ** p<0.0001.

Article Snippet: Cells were passed through a 70 μm strainer and stained at 4 °C for 30 minutes with following antibodies (1:100): SiglecF-APC (catalog number 50–1702-82, clone 1RNM44N, eBioscience), MHCII-APC-Cy7 (catalog number 47–5321-82, clone M5/114.15.2, eBioscience), and CD11c-PE (catalog number 557401, clone HL3, BD Biosciences).

Techniques: Microarray, RNA Sequencing, Immunofluorescence, Control, Quantitation Assay, Imaging

Journal: Nature immunology

Article Title: Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage

doi: 10.1038/s41590-018-0276-y

Figure Lengend Snippet: a, Pdgfa expression in lung scRNA-seq macrophage clusters and by bulk RNA-seq of SiglecF + CD11c + lung macrophages after bleomycin injury. Mean is marked. Wald test 2-sided p-value is presented (for the lung scRNA-seq data, n=3 biologically independent mice for bleomycin-injured and n=6 biologically independent mice for healthy control; for the bulk RNA-seq data, n=2 biologically independent mice for baseline, n=3 biologically independent mice for all other conditions). b, Lung immunofluorescence at baseline (bottom) and 14 days after injury (top) in tamoxifen-induced Cx3cr1 -CreERT2 / Rosa26 loxp STOP loxp -TdTomato mice (representative images are shown; quantitation is for the 14-day time point in n=3 biologically independent mice, 5 images per mouse). Scale bar, 50 μm. c, 3T3 fibroblast gap closure in response to conditioned media (CM) from lung macrophages sorted by MHCII expression, with and without Pdgf-aa blocking antibody (Ab; n=8 biologically independent mice total, each point a separate assay). d , EDU quantitation in 3T3 fibroblasts co-cultured with TdTomato + cells sorted from tamoxifen-induced Cx3cr1 -CreERT2 / Rosa26-loxp-STOP-loxp-TdTomato mice 14 days after injury. Mean is marked (n=3 biologically independent co-cultures). e , t-SNE of SingleR-annotated fibroblasts from the lung scRNA-seq dataset showing intensity of cell cycle signature (n=3 biologically independent mice for bleomycin, n=6 biologically independent mice for control). Box plot center lines are median, box limits are upper and lower quartiles, and whiskers denote the largest and smallest values no more than 1.5 times the interquartile range from the limits. Wilcoxon test 2-sided p-values are presented. * p < 0.01; ** p < 0.0001.

Article Snippet: Cells were passed through a 70 μm strainer and stained at 4 °C for 30 minutes with following antibodies (1:100): SiglecF-APC (catalog number 50–1702-82, clone 1RNM44N, eBioscience), MHCII-APC-Cy7 (catalog number 47–5321-82, clone M5/114.15.2, eBioscience), and CD11c-PE (catalog number 557401, clone HL3, BD Biosciences).

Techniques: Expressing, RNA Sequencing, Control, Immunofluorescence, Quantitation Assay, Blocking Assay, Cell Culture

Journal: Journal of biomedical materials research. Part A

Article Title: N -dihydrogalactochitosan as a potent immune activator for dendritic cells

doi: 10.1002/jbm.a.35991

Figure Lengend Snippet: Determining the optimum concentration of GC by flow cytometry. Relatively immature DC2.4 cells were cultured in the absence and the presence of 0.01, 0.1, and 1 mg/mL GC and then were analyzed by flow cytometry gating out dead cells. (Panels A and B) cells were unstained and stained with PE-Cy7 conjugated-rat IgG2b K isotype control as background control, (panels C–E) cells were incubated with 0.01, 0.1, and 1 mg/mL GC and then stained with PE-Cy7 conjugated MHC Class II (I-A/I-E) antibody (M5/114.15.2) and (F) Histogram showing MHC class II expression level (means + SD) for treated DC2.4 as described above. Experiments were repeated three times.

Article Snippet: PE-Cy7 conjugated MHC Class II (I-A/I-E) antibody (M5/114.15.2), FITC conjugated CD80/B7-1 antibody (16-10A1) and its FITC conjugate isotype control antibody Hamster IgG, PE-Cy7 conjugated Integrin alpha X/CD11c antibody, CD16 + CD32 antibody for blocking unspecific binding were all bought from ThermoFisher Scientific.

Techniques: Concentration Assay, Flow Cytometry, Cell Culture, Staining, Control, Incubation, Expressing

Journal: Journal of biomedical materials research. Part A

Article Title: N -dihydrogalactochitosan as a potent immune activator for dendritic cells

doi: 10.1002/jbm.a.35991

Figure Lengend Snippet: Confocal imaging for expression of activation markers showing DC2.4 cells after 24 hours of incubation. FITC-conjugated ovalbumin (green) uptake (panels A–C); MHC class II expression by PE-CY7 conjugated MHC Class II (I-A/I-E) antibody (red) (M5/114.15.2) (panels D–F); CD11c expression by PE-CY7 conjugated integrin alpha X/CD11c antibody (red) (panels G–I); in control DC2.4 cells (panels A, D, G); DC2.4 incubated with 1 mg/mL GC for 24 h (Panels B, E, H); DC2.4 incubated with 50 μg LPS for 24 h. Blue staining is Hoechst nuclear stain. Scale bar = 10 μm.

Article Snippet: PE-Cy7 conjugated MHC Class II (I-A/I-E) antibody (M5/114.15.2), FITC conjugated CD80/B7-1 antibody (16-10A1) and its FITC conjugate isotype control antibody Hamster IgG, PE-Cy7 conjugated Integrin alpha X/CD11c antibody, CD16 + CD32 antibody for blocking unspecific binding were all bought from ThermoFisher Scientific.

Techniques: Imaging, Expressing, Activation Assay, Incubation, Control, Staining