mouse il 1ra il 1f3 duoset kit  (R&D Systems)

Journal: PLOS Pathogens

Article Title: Single-cell transcriptomics unveils skin cell specific antifungal immune responses and IL-1Ra- IL-1R immune evasion strategies of emerging fungal pathogen Candida auris

doi: 10.1371/journal.ppat.1012699

Figure Lengend Snippet: (A) Heatmap represents the expression of Il1rn in the cell types (y-axis) between the infected and uninfected groups. The normalized gene counts were plotted in the heatmap, and the scale indicates red for high, blue for low, and white for moderate expression in the samples. Each column represents a different sample. (B) The violin plot depicts the scaled count of Il1rn in myeloid subsets from both uninfected and infected samples. The scaled count were normalized using SCTransform method. IL-1Ra levels in the (C) skin and (D) serum of mice after 3 DPI of C . auris AR0387 or C . albicans SC5314 ( n = 8–10 mice/group). Uninfected mice received 100 μL of 1X PBS on the day of infection. (E) Fungal burden in the skin tissue of indicated mice on 3 DPI of C . auris AR0387 or C . albicans SC5314 ( n = 8–10 mice/group). Uninfected mice received 100 μL of 1X PBS on the day of infection. (F) The bar graph represents the neutrophil-killing activity of C . auris AR0387 in the presence and absence of recombinant IL-1Ra. (G) Skin fungal burden of the mice groups injected with neutralizing anti-mouse IL-1Ra monoclonal antibody or 100 μl of 1X PBS after 3 DPI of C . auris AR0387 in the murine skin. ( n = 3–5 mice/group) (H-K) Quantification of IL-1Ra level and fungal burden in mice injected with anti-Ly6G antibody and Rat IgG2a isotype control (Isotype) for neutrophil depletion after 2 DPI of C . auris AR0387 ( n = 6–8 mice/group). H) Representative flow plots and I) percentage and absolute number of CD11b + Ly6G + neutrophils in the infected skin tissue of mice injected with anti-Ly6G antibody or Rat IgG2a isotype control (Isotype) antibody. (J) IL-1Ra level and (K) CFU were determined from the homogenate of infected skin tissue. (L-O) Measurement of IL-1Ra production and fungal burden in clodrosome (C.L) and 1X PBS (Ctrl) injected mice groups for macrophage depletion after 2 DPI of C . auris AR0387 ( n = 9–10 mice/group). (L) Representative flow plots of F4/80+ MHCII+ macrophage in the infected skin tissue of mice injected with clodrosome (C.L) or 1X PBS (Ctrl) injected mice groups. (M) IL-1Ra level and (N) CFU determined from the homogenate of infected skin tissue from 2 DPI of C . auris 0387 (n = 12 mice/group).(O) IL-1Ra level and (P) CFU determined from the homogenate of infected skin tissue from 12 DPI of C . auris 0387 (n = 8–10 mice/group). Error bars represent mean ± SEM. * p < 0.05, ** p <0.01, *** p <0.001, **** p <0.0001, NS, non-significant. Statistical significances were calculated using Mann–Whitney U. Abbreviations–DPI, days post-infection; C.L, clodrosome.

Article Snippet: IL-1Ra production in skin homogenate, culture supernatant, and serum was quantified using a mouse IL-1Ra/IL-1F3 DuoSet kit (RnD Systems) by following the manufacturer’s standard operating protocol.

Techniques: Expressing, Infection, Activity Assay, Recombinant, Injection, Control, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Single-cell transcriptomics unveils skin cell specific antifungal immune responses and IL-1Ra- IL-1R immune evasion strategies of emerging fungal pathogen Candida auris

doi: 10.1371/journal.ppat.1012699

Figure Lengend Snippet: (A) The representative flow plots represents the percentage of CD11b + Ly6G + neutrophils and CD64 + MHCII + macrophage in the WT and Il1r1 -/- mice skin tissue infected with C . auris 0387. The bar graph represents the percentage and absolute number of CD11b + Ly6G + neutrophils after (B) 3 DPI and (C) 12 DPI and the CD64 + MHCII + macrophage after (D) 3 DPI and (E) 12 DPI in the WT and Il1r1 -/- mice skin tissue infected with C . auris 0387 ( n = 4–8 mice/group). (F) The representative flow plots represents the percentage of CD11b + Ly6G + neutrophils and CD64 + MHCII + macrophage in the WT and Il1r1 -/- mice skin tissue infected with 0387 pmr1Δ . The bar graph represents the percentage and absolute number of CD11b + Ly6G + neutrophils after (G) 3 DPI and (H) 12 DPI and the CD64 + MHCII + macrophage after (I) 3 DPI and (J) 12 DPI in the WT and Il1r1 -/- mice skin tissue infected with 0387 pmr1Δ ( n = 5–10 mice/group). For C . auris 0387 pmr1Δ infection 5 × 10 6 CFU per mice were used. (K) The bar graph represents the fungal survival of C . auris 0387 and 0387 pmr1Δ primed with neutrophils isolated from bone marrow of WT and Il1r1 -/- mice. ( n = 6) (L) Measurement of IL-1Ra levels from the culture supernatant of BMDM uninfected or infected with live C . auris 0387 or 0387 pmr1Δ for 16 h. ( n = 12). Error bars represent mean ± SEM. * p < 0.05, ** p <0.01, *** p <0.001, **** p <0.0001, NS, non-significant. Abbreviations—DPI, days post-infection; WT, wild type; BMDM, bone marrow-derived macrophages. Statistical significances were calculated using Mann–Whitney U.

Article Snippet: IL-1Ra production in skin homogenate, culture supernatant, and serum was quantified using a mouse IL-1Ra/IL-1F3 DuoSet kit (RnD Systems) by following the manufacturer’s standard operating protocol.

Techniques: Infection, Isolation, Derivative Assay, MANN-WHITNEY

Journal: Nutrients

Article Title: Short-Term Caloric Restriction and Subsequent Re-Feeding Compromise Liver Health and Associated Lipid Mediator Signaling in Aged Mice.

doi: 10.3390/nu15163660

Figure Lengend Snippet: Figure 1. Impact of aging on the hepatic proteome and inflammatory microenvironment of mice with ad libitum access to food. (A) Average body weight of young (Y) and old (O) mice over the course of the experiment (n = 5). (B) Principal component analysis of the hepatic proteome. The mean for each experimental cohort is indicated by a rhombus; single data are given as circles. (C) Volcano plot of changes to the hepatic proteome of old mice in comparison to young animals (Table S1, Y: n = 6; O: n = 4). Dashed lines indicate the cut-off for significance (q < 0.05) and absolute fold change (log2 > 0.58). (D) The relative median abundance of proteomic markers of innate immune cell subtypes in the liver (HC—hematopoietic cells, Mφ—macrophages, MDM—monocyte-derived macrophages, TRM—tissue-resident macrophages, M1—classically activated (M1) macrophages, M2—alternatively activated (M2) macrophages). Data points at baseline = not detectable (Table S1, Y: n = 5–6; O: n = 3–4). (E) Cytokine levels in whole liver lysates were measured by ELISA (n = 6). (F) Hepatic MDA levels in Y and O mice (n = 6). (G) Relative protein abundance of biomarkers for liver inflammation [26]: glutathione S-transferase P1 (GSTP1), glutathione peroxidase 3 (GPX3), protein S100A11, peptidyl-prolyl cis-trans isomerase (FKBP1A), PDZ and LIM domain protein 1 (PDLIM1) (Table S1, Y: n = 6; O: n = 4). Statistics: Data are shown as (A,E,G) mean ± SEM, (C,D) median or (F) median (min to max); p- or q-values were calculated by one-way ANOVA for multiple comparisons with Tukey’s posthoc test or Brown–Forsythe and Welch ANOVA with Dun- nett’s T3 posthoc test (Table S3) or Spectronaut™(Table S1). ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001, ns = not significant.

Article Snippet: Whole liver lysates were used to determine cytokine and chemokine levels using R&D Systems ELISA Kits for IL-1β (DY401-05), IL-1ra (DY480-05), IL-6 (DY406-05), IL10 (DY417-05), TNF-α (DY410-05) (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Derivative Assay, Enzyme-linked Immunosorbent Assay, Quantitative Proteomics

Journal: Nutrients

Article Title: Short-Term Caloric Restriction and Subsequent Re-Feeding Compromise Liver Health and Associated Lipid Mediator Signaling in Aged Mice.

doi: 10.3390/nu15163660

Figure Lengend Snippet: Figure 3. Impact of CR and RF intervention on the hepatic proteome and inflammatory microenvi- ronment. (A) Average body weight of old (O), caloric restricted (CR), and re-fed (CR+RF) mice over the course of the experiment. Values are presented as mean ± SEM (n = 5). (B) Principal component analysis of the hepatic proteome of young (Y), O, CR, CR+RF mice. The mean for each experimental group is indicated by rhombus; single values are given as circles. Volcano plots of changes to the hepatic proteome (C) in CR mice and in CR+RF mice compared to old mice (Table S1, O: n = 4; CR, CR+RF: n = 6). Dashed lines indicate the cut-off for significance (q < 0.05) and absolute fold change (log2 > 0.58). (D) Relative protein abundance of biomarkers for liver inflammation [26]. Heatmap represents the log2-fold change compared to the median in young mice (Table S1, O: n = 4; CR, CR+RF: n = 6). (E) Relative abundance of proteomic markers of innate immune cell subtypes in the liver (HC—hematopoietic cells, Mφ—macrophages, MDM—monocyte-derived macrophages, TRM—tissue-resident macrophages, M1—classically-activated (M1) macrophages, M2—alternatively activated (M2) macrophages). Data points at baseline were not detectable (Table S1, O: n = 3–4; CR, CR+RF: n = 5–6). (F) Cytokine levels in whole liver lysates were assessed by ELISA (n = 6). The dotted line represents the mean level in young mice (Figure 1E). (G) Hepatic MDA levels in O, CR, CR+RF mice (n = 6). The dotted line represents the mean level in young mice (Figure 1F). Statistics: Data are shown as (A,D,F) mean ± SEM, (C,E) median or (G) median (min to max); p- and q-values were calculated by one-way ANOVA for multiple comparisons with Tukey’s posthoc test or Brown–Forsythe and Welch ANOVA with Dunnett’s T3 posthoc test (Table S3) or Spectronaut™ (Table S1). Statistical significance is indicated by asterisks for comparisons between CR and O, by hashes for comparisons between CR+RF and O and by }for comparisons with the levels of young mice. */# p ≤0.05, **/}}p ≤0.01, ns = not significant.

Article Snippet: Whole liver lysates were used to determine cytokine and chemokine levels using R&D Systems ELISA Kits for IL-1β (DY401-05), IL-1ra (DY480-05), IL-6 (DY406-05), IL10 (DY417-05), TNF-α (DY410-05) (R&D Systems, Minneapolis, MN, USA).

Techniques: Quantitative Proteomics, Derivative Assay, Enzyme-linked Immunosorbent Assay