Journal: Journal of Leukocyte Biology

Article Title: Lymph node stromal cells vary in susceptibility to infection but can support the intracellular growth of Listeria monocytogenes

doi: 10.1093/jleuko/qiae040

Figure Lengend Snippet: SVEC4–10 cells were infected with L. monocytogenes (Lm) at MOI=10. (A-B) Mean percentage (analyzed by Mann-Whitney) of Lm SD2000 inoculum that was gentamicin-resistant 1 hr post-infection (hpi) in (A) SVEC4–10 cells or (B) Caco-2 cells. (C) Intracellular growth assay using stationary phase Lm SD2000. For panels A and C, mean values (±SEM) for triplicate samples from one of two experiments is shown. For panel B, mean values (±SEM) for quadruplet samples from one experiments is shown. (D) Representative images for differential “in/out” staining of cells infected with stationary phase Lm SD2710 (constitutive GFP), fixed at 4 hpi, and stained with DAPI (nucleus/blue), and anti-Listeria antibody (Lm/red). Scale bar, 5 μm. Dashed line indicates the outline of infected cell from the brightfield image. (E) Stacked bar indicates the percentage of Lm-infected cells (n=300) with only intracellular bacteria (IC), both intracellular and extracellular (IC+ EC), or only extracellular bacteria (EC). (F) Violin plot indicates the number of intracellular Lm observed per cell (n=300). For panels C, D and E data from one of two independent experiments are shown. (G) Representative image for phalloidin staining of SVEC4–10 cells infected with Lm SD2710 at 4 hpi; white arrows indicate Lm associated with actin; yellow arrowhead indicates Lm not associated with actin. Scale bar, 10 μm. Two independent experiments (n=100 cells analyzed in each) were performed; in Exp. 1, 72% of Lm were associated with actin and in Exp. 2 80% were. (H) Representative images from one of three independent plaque assays using SVEC4–10 cells or L2 fibroblasts infected with Lm SD2000 and visualized 4 dpi.

Article Snippet: Permeabilized cells were incubated with goat anti-rabbit IgG-Texas Red-X phalloidin (ThermoFisher) diluted 1:100 in TBS+BSA for 30 min at room temperature.

Techniques: Infection, MANN-WHITNEY, Growth Assay, Staining, Bacteria

Journal: Journal of Leukocyte Biology

Article Title: Lymph node stromal cells vary in susceptibility to infection but can support the intracellular growth of Listeria monocytogenes

doi: 10.1093/jleuko/qiae040

Figure Lengend Snippet: LNSC were sort-purified from the MLN and PLN of uninfected BALBc/ByJ mice and infected with Lm at an MOI of 10. (A) Gentamicin protection assay for cells infected with Lm SD2000; pooled data from eight separate experiments (each designated by a different symbol type) were analyzed by ANOVA with Tukeys multiple comparison test. In each experiment, total cells sorted from three mice were used at two time points. (B) Representative images for differential ”in/out” staining of sort-purified LNSC infected with Lm SD2710 (GFP+), fixed at 8 hpi and stained with DAPI (nuclei; blue), and anti-Lm antibody (red); scale bar, 5 μm. (C) Symbols indicate the number of intracellular Lm observed in a single infected cell; horizontal lines indicate median values for n=100 cells. For panels B and C, data from one of two independent experiments are shown. (D) Representative images for phalloidin staining of cells infected with Lm SD2710. White arrows indicate actin tails associated with Lm; yellow arrowheads indicate Lm not associated with actin. Scale bar, 5 μm. (E) Mean percent (±SEM) of intracellular Lm associated with actin. Each symbol represents results from a single experiment. (F) IFNβ present in supernatants of uninfected (U) and Lm SD2000-infected (Lm) cells (3 × 104 cells/well) at 16 hours was measured by multiplex immunoassay; symbols indicate mean values (+/− SEM) for duplicate samples from three separate experiments and were analyzed by one-way ANOVA. (G) Fold change of cytokine responses measured by multiplex immunoassay in Lm-infected cells relative to uninfected cells. Dashed lines indicate limits of detection.

Article Snippet: Permeabilized cells were incubated with goat anti-rabbit IgG-Texas Red-X phalloidin (ThermoFisher) diluted 1:100 in TBS+BSA for 30 min at room temperature.

Techniques: Purification, Infection, Comparison, Staining, Multiplex Assay

Journal: Neural Regeneration Research

Article Title: Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming

doi: 10.4103/NRR.NRR-D-23-01299

Figure Lengend Snippet: PFP-OLNDs treatment alleviates TH + neuron death in the MPTP model of PD. (A, B) Representative immunofluorescence staining for Iba1 (red, Cy3) in the substantia nigra 4 days after the last MPTP injection. In vivo intraperitoneal injections of MPTP in mice greatly increased the density of microglia in the substantia nigra region, and intranigral PFP-OLNDs injections prevented the MPTP-induced microglia activation. (C, D) Representative immunofluorescence staining for TH (green, Alexa Fluor 488) in the substantia nigra 4 days after the last MPTP injection. Intranigral PFP-OLNDs injections reduced TH + neuronal death induced by MPTP treatment. Scale bars: 100 μm (upper panel); 50 μm (lower panel). Data are expressed as mean ± SEM ( n = 6 mice per group). * P < 0.05, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Bonferroni post hoc test). Iba1: Ionized calcium binding adaptor molecule 1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson’s disease; PFP-OLNDs: perfluoropentane-based oxygen-loaded nanodroplets; TH: tyrosine hydroxylase.

Article Snippet: After washing, the slices were incubated for 1 hour at room temperature with Cy3-conjugated goat anti-rabbit IgG (1:300, Cat# GB21303, RRID AB_2861435, Servicebio, Wuhan, China) and goat anti-mouse IgG labeled with Alexa Fluor 488 (1:400, Cat# GB25301, RRID: AB_2904018, Servicebio).

Techniques: Immunofluorescence, Staining, Injection, In Vivo, Activation Assay, Binding Assay