galectin 3 mcherry  (Galectin Therapeutics)

Journal: Nature Communications

Article Title: The transcription factor Zeb1 controls homeostasis and function of type 1 conventional dendritic cells

doi: 10.1038/s41467-023-42428-7

Figure Lengend Snippet: a WT or Zeb1-deficient Flt3L-cDC1, or Zeb1-deficient Flt3L-cDC1 lentivirally transduced with Cybb containing synonymously mutated miR-96 binding sites were cultured for 3 days with CFSE-labeled OT-I T cells and different dose of HKLM-OVA and assayed for OT-I proliferation and activation (CFSE - CD44 + ). b WT Flt3L-cDC1 retrovirally transduced with empty vector (EV) or miR-96 or miR-182 were cultured and analyzed as described in ( a ), with various dose of HKLM-OVA as antigens. c Intracellular and mitochondrial ROS production in WT and Zeb1-deficient Flt3L-cDC1 challenged with HKLM-OVA for indicated times, were measured by flow cytometric analysis of CellROX and MitoSOX fluorescence. The mean fluorescent intensity (MFI) of CellROX and MitoSOX fluorescence were quantified at the bottom. d , e Phagosomal ROS production in WT and Zeb1-deficient Flt3L-cDC1 exposed with OxyBURST/Alexa Fluor 647-conjugated HKLM-OVA for indicated times, were measured by flow cytometric analysis ( d ) of OxyBURST and Alexa Fluor 647 fluorescence. The phagosomal ROS production was quantified as the ratio of OxyBURST + cells to Alexa Fluor 647 + cells ( e , top row) or MFI of OxyBURST in Alexa Fluor 647 + cells ( e , bottom row). f WT and Zeb1-deficient Flt3L-cDC1 were cultured with HKLM-OVA for 30 min, and treated with or without DPI (10 μM) for 4 h before addition of CFSE-labeled OT-I T cells, and assayed for OT-I proliferation and activation (CFSE - CD44 + ). g , h Confocal microscope images of mCherry::galectin-3-expressed WT and Zeb1-deficient Flt3L-cDC1 challenged with Alexa Fluor 647-labeled HKLM-OVA. Colocalizing signal of Galectin-3 + HKLM + cells were counted and plotted as a ratio of total HKLM + cells. Each symbol represents an individual sample, small horizontal lines indicate the mean (±s.d.). Data are representative of three independent experiments. Data are presented as mean ± s.d. Statistical analysis was performed using Two-tailed unpaired Student’s test ( h ) or two-way ANOVA with Tukey’s ( a , f ) or Sidak’s ( b , e ) multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: The expression plasmid for mCherry:Galectin-3 (Cat#85662) was purchased from Addgene.

Techniques: Transduction, Binding Assay, Cell Culture, Labeling, Activation Assay, Plasmid Preparation, Fluorescence, Microscopy, Two Tailed Test

Journal: Immunology

Article Title: Pneumolysin suppresses the initial macrophage pro‐inflammatory response to Streptococcus pneumoniae

doi: 10.1111/imm.13546

Figure Lengend Snippet: Effects of ply on Streptococcus pneumoniae growth, phagocytosis, cell death, and phagosome integrity in the MDM infection model. (a and b) S. pneumoniae growth in the presence of and uptake by macrophages is unaffected by ply. MDM were incubated with TIGR4 (black) or TIGR4Δ ply (grey) at MOI 10 and supernatant cultured after 6 h plated to determine (a) bacterial CFU in the supernanant, and (b) determine adherent and internalized bacteria as a percentage of the inoculum using an antibiotic protection assay. (c) MDM were incubated with TIGR4 or TIGR4Δ ply at MOI 10 for 6 h, then stained with propidium iodide as a marker of loss of nuclear integrity. The proportion of MDM that stained positive were measured using Hermes high‐content imaging system (magnification ×10) and automated image analysis. Data are shown as means ± SEM of three experiments and analysed by one‐way ANOVA with Tukey's multiple comparisons test. (d and e) Effects of inhibition of necroptosis; 100 μM Necrostain (N0164, sigma) was added for 1 h to MDM before infection with TIGR4 or TIGR4Δ ply (MOI 10) for 6 h. (d) Cell viability was assessed by measuring supernatant LDH levels, and (e) supernatant TNF levels measured using ELISAs. Data from four experiments are presented means ± SEM and analysed by two‐way ANOVA with Sidak multiple comparisons test. (f and g) Confocal microscopy for localisation of FAM‐SE labelled TIGR4 and TIGR4Δ ply (green) after incubation with THP‐1 cells expressing mCherry galectin 3 (red, MOI 100). Images were acquired after quenching fluorescence from external bacteria with trypan blue. The proportion of galectin 3 positive bacteria were measured by microscopy. Sample images are shown in panel F demonstrating that TIGR4 bacteria colocalise with galactin 3, whereas TIGR4Δ ply do not. The proportion of galectin 3 positive bacteria for TIGR4 and TIGR4Δ ply are shown in panel (g) as means ± SEM from four experiments.

Article Snippet: For the galectin 3 phagosome assay THP1 cells expressing mCherry Galectin 3 were differentiated with 25 ng/ml PMA then 200 μg/ml gentamicin was added.

Techniques: Infection, Incubation, Cell Culture, Staining, Marker, Imaging, Inhibition, Confocal Microscopy, Expressing, Fluorescence, Microscopy