Journal: Gut Microbes

Article Title: Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

doi: 10.1080/19490976.2022.2127455

Figure Lengend Snippet: Phenotypic and functional characterisation of MDSCs. MDSCs were differentiated from bone marrow of DA rats in the presence of FLT3/GM-CSF/IL-6 (MDSCs, blue) or FLT3/GM-CSF/IL-6 and PGE2 (MDSC-PGE2, green) for 4 days. A-C . Representative flow cytometry histograms are showing the expression of indicated markers from one experiment, out of three with similar results (for summarised results see Supplementary Figure 1). Common markers expressed by rat MDSCs (CD11b, SIRP-α, HIS48) ( A ); myeloid lineage markers (OX62, CD68 and RP1) ( B ); functional markers (MHCII, CD86 and CD80) are shown ( C ). D . The proliferation of ConA-stimulated Cell Trace Far Red-labelled splenocytes (3 × 10 5 /well) is shown, in which the splenocytes were cultivated without (wo) MDSCs (dashed line) or in the presence of MDSCs or MDSC-PGE2 (each at 6 × 10 4 , 3 × 10 4 , 1.5 × 10 4 or 0.75 × 10 4 /well, providing 1:5–1:40 MDSCs: splenocytes ratio, respectively) for 4 days. A representative analysis of 1:10 and 1:40 MDSC/splenocyte ratios is shown along with summarised data. E . Relative mRNA levels of COX-2, iNOS, ARG1 are shown relative to β-actin expression, as well as NO production as detected by Griess reaction method. D-E . The summarized data is shown as mean ± SD of three independent experiments. Student’s t -test was used for comparison (* p < 0.05).

Article Snippet: Intracellular staining of cells was conducted after surface staining using a flow cytometry fixation and permeabilization kit I (R&D Systems).

Techniques: Functional Assay, Flow Cytometry, Expressing, Comparison

Journal: Gut Microbes

Article Title: Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

doi: 10.1080/19490976.2022.2127455

Figure Lengend Snippet: Immune response in animals treated with MDSCs and MDSC-PGE2 at the peak of EAE. A . The percentages of interferon (IFN)-γ-producing NK (CD161 + IFN-γ + ), Th17 (CD4 + IL-17 + ) and (IFN)-γ-producing T (CD3 + IFN-γ + ) lymphocytes in the spleen and spinal cord samples are shown, after their analysis upon isolation from animals (n = 5 animals in each group) at the peak of EAE (15dpi) in the control (ctl_EAE, red) group, and groups treated with either MDSCs (MDSC_EAE, blue) or MDSC-PGE2 (MDSC-PGE2_EAE, green). Data was obtained by flow cytometry analysis as shown in Supplementary Figure 4. The percentages of regulatory T cells (Tregs) (CD25 + FoxP3 + ) and IL-10-producing Tregs (CD25 + FoxP3 + IL-10 + ) were shown from the total gated CD4 + lymphocytes (the gating strategy is shown in Supplementary Figure 5). The results on total cell numbers are shown in Supplementary Figure 6. B . The levels of cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-4, IL-17, IFN-γ and IL-10) were measured from ex vivo splenocytes isolated from each animal (as in A ), and cultivated (2 × 10 6 /mL/well of 24-wells plate) for 24 h in the complete RPMI medium in the presence of ConA. C . The relative mRNA levels of claudin and occludin in intestinal samples of animals treated as in (A ), were calculated relative to β-actin and presented as fold change expression relative to healthy animals D . The activity of alkaline phosphatase in faecal material collected at the peak of EAE, as well as from healthy animals were analysed as a measure of intestinal barrier integrity. A-D . Data is shown as mean ± SD (n = 5) from one experiment, out of two with similar results. One-way ANOVA followed by Tukey post hoc test was used for comparison (* p < 0.05, ** p < 0.01) to control EAE (ctl_EAE).

Article Snippet: Intracellular staining of cells was conducted after surface staining using a flow cytometry fixation and permeabilization kit I (R&D Systems).

Techniques: Isolation, Control, Flow Cytometry, Ex Vivo, Expressing, Activity Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: Estrogens Modulate Somatostatin Receptors Expression and Synergize With the Somatostatin Analog Pasireotide in Prostate Cells

doi: 10.3389/fphar.2019.00028

Figure Lengend Snippet: Effect of estradiol on cell cycle. Flow cytometry analysis of propidium iodide-labeled EPN (A) or CPEC (B) cells was done, as described in Methods. Quiescent cells were left under basal conditions or treated with estradiol (20 nM; E 2 ) for the indicated times. Histograms represent the % counts/FL2 areas of labeled cells in the different cell cycle phases after 24 or 48 h of hormonal treatment. Means and SEMs are shown. n represents the number of experiments. ∗ p < 0,05 for each experimental point vs. the corresponding untreated control.

Article Snippet: Permeabilization was performed by incubating 10 6 cells in 0,5 ml of flow cytometry analysis (FACS) permeabilizing solution, which contains RNAses and propidium iodide (Beckton Dickinson).

Techniques: Flow Cytometry, Labeling, Control

Journal: Frontiers in Pharmacology

Article Title: Estrogens Modulate Somatostatin Receptors Expression and Synergize With the Somatostatin Analog Pasireotide in Prostate Cells

doi: 10.3389/fphar.2019.00028

Figure Lengend Snippet: Effect of both estradiol and pasireotide on cell cycle. Flow cytometry analysis of propidium iodide-labelled EPN (A) or CPEC (B) was done, as described in Methods. In panel (A,B) , quiescent cells were left under untreated or treated for 24 h with 0,1 μM pasireotide, in the absence or presence of 20 nM estradiol. Histograms in panel (A,B) represent the % counts/FL2 areas of labeled cells in the different cell cycle phases after the indicated treatments. Means and SEMs are shown. n represents the number of experiments. ∗ p < 0,05; ∗∗ p < 0,01. In panel (C) , quiescent EPN (left section) or CPEC (right section) cells were left untreated or treated for 24 h with 0,1 μM pasireotide, in the absence or presence of 20 nM estradiol. Lysate proteins were separated by SDS-PAGE, transferred to PDVF membrane and filters were then analyzed by Western blot, using the anti-activated caspase 3 or anti-tubulin antibodies.

Article Snippet: Permeabilization was performed by incubating 10 6 cells in 0,5 ml of flow cytometry analysis (FACS) permeabilizing solution, which contains RNAses and propidium iodide (Beckton Dickinson).

Techniques: Flow Cytometry, Labeling, SDS Page, Membrane, Western Blot

Journal:

Article Title: Female X-chromosome mosaicism for gp91phox expression diversifies leukocyte responses during endotoxemia

doi: 10.1097/CCM.0b013e3181eb9ed6

Figure Lengend Snippet: Lipopolysaccharide (LPS)-induced changes in bone marrow (BM) and blood cell composition in WT, gp91phox-deficient and mosaic animals. Twenty-four hours after LPS or saline controls, BM cells and blood were collected and processed for flow cytometry analyses, as described in Materials and Methods. Graphs A and B depict BM myeloid cell content identified by CD45/CD11b double positive (A) and B-cell content identified by CD45/CD19 double positive staining (B). Graphs C–F depict the numbers of circulating CD11b+ (mostly neutrophils) and different T-cell (CD4+ and CD8+) and B-cell (CD19+) subsets as indicated. Cell numbers were calculated from percent distribution of cells and total white blood cell yield. Statistically significant difference (p < .05): *compared with control within the same genotype; #compared with WT or heterozygous controls. Mean ± SEM, n = 7–8 animals in each group.

Article Snippet: Flourochrome conjugated antibodies, assay diluents, lysing and permeabilizing flow cytometry solutions and kits were purchased from BD Biosiences (San Jose, CA) and BD Pharmingen (San Diego, CA).

Techniques: Flow Cytometry, Staining