mouse anti marco  (Bio-Rad)

Journal: Cell reports

Article Title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis

doi: 10.1016/j.celrep.2024.113894

Figure Lengend Snippet: (A) scRNA-seq UMAP feature plots for transcription factor TCF7L2 motif enrichment and gene expression. Numbers indicate different cell clusters, as outlined in . (B) RT-qPCR for key exhaustion genes in BMMCs under PBS control or repetitive LPS stimulation in the presence of different concentrations of Wnt agonist 1. Expression levels were normalized to the geometric mean of Ube2l3 , Oaz1 , and Nktr (mean expression ± SD; n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test; ****p-adj < 0.0001; ***p-adj < 0.001; **p-adj < 0.01; *p-adj < 0.05; ns, not significant; differences are not significant unless otherwise specified; see for exact p values). (C) Flow cytometry mean fluorescence intensity (MFI) for exhaustion (CD38, MARCO, PD-L1, CXCR2) and macrophage (F4/80) markers relative to PBS control. Boxplots indicate median MFI values (n = 5–7; one-way ANOVA with Sidak’s multiple comparisons test). (D) Gating strategy (top) and population statistics (bottom) for non-classical (Ly6C low ), intermediate (Ly6C int ), and classical (Ly6C high ) monocyte subtypes in cultured BMMCs (n = 6–13; one-way ANOVA with Sidak’s multiple comparisons test). (E) NAD + levels in cultured BMMCs normalized to total protein levels (n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test). (F) Correlation heatmap for average DNA methylation levels at key exhaustion loci in cultured BMMCs (n = 3–9 for each condition). (G) UCSC browser track views of average DNA methylation at the Plac8 promoter (chr5: 100,572,230–100,572,607), Cebpa/g distal enhancer (chr7: 35,064,805–35,065,304), and Tcf7l2 intron (chr19: 55,768,986–55,769,585). ENCODE annotated promoters (red) and enhancers (orange) are depicted in the bottom track.

Article Snippet: For Panel 2, cells were stained with antibodies against Ly6C (Pacific Blue; Biolegend #128014), CD11b (BV650; BD Biosciences #563402), Ly6g (PE-Cy5.5; Elabscience #E-AB-F1108I), CXCR2 (Alexa Fluor 488; R&D Systems # FAB2164G), CD68 (APC-Fire750; Biolegend #137041), CD172a (BV510; BD Biosciences #740159), CX3CR1 (PE-Cy7; Biolegend #149016), F4/80 (BV711; BD Biosciences #565612), CD38 (BV750; BD Biosciences #747103), PD-L1 (BV421; BD Biosciences #564716), MARCO (Alexa Fluor 647; R&D Systems #FAB29561R), FAS (BV605; Biolegend #152612), MHCII (PerCP; Biolegend #107624), CD168 (PE; Novus #NBP1–76538PE), and TREM2 (Alexa Fluor 700; R&D Systems #FAB17291N).

Techniques: Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, Cell Culture, DNA Methylation Assay

Journal: Cell reports

Article Title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis

doi: 10.1016/j.celrep.2024.113894

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For Panel 2, cells were stained with antibodies against Ly6C (Pacific Blue; Biolegend #128014), CD11b (BV650; BD Biosciences #563402), Ly6g (PE-Cy5.5; Elabscience #E-AB-F1108I), CXCR2 (Alexa Fluor 488; R&D Systems # FAB2164G), CD68 (APC-Fire750; Biolegend #137041), CD172a (BV510; BD Biosciences #740159), CX3CR1 (PE-Cy7; Biolegend #149016), F4/80 (BV711; BD Biosciences #565612), CD38 (BV750; BD Biosciences #747103), PD-L1 (BV421; BD Biosciences #564716), MARCO (Alexa Fluor 647; R&D Systems #FAB29561R), FAS (BV605; Biolegend #152612), MHCII (PerCP; Biolegend #107624), CD168 (PE; Novus #NBP1–76538PE), and TREM2 (Alexa Fluor 700; R&D Systems #FAB17291N).

Techniques: Blocking Assay, Negative Control, Recombinant, Methylation, SYBR Green Assay, DNA Methylation Assay, Software, Isolation, Reverse Transcription, Multiplex Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: AXL promotes lymphangiogenesis by amplifying VEGF-C-mediated AKT pathway

doi: 10.1007/s00018-024-05542-3

Figure Lengend Snippet: AXL is expressed by MARCO + LECs in murine LN. A. Representative images of naive murine lymph node immunostained for AXL (red), LYVE-1 (green), MARCO (white). B-D . In vivo Tumor-associated lymphangiogenesis assay. The experimental design of the assay is given in ( B ). Gelatin sponges containing (tumor draining: Td) or not (sham) melanoma cells were implanted in mice ear. The sentinel LN (Td LN or sham LN) were resected 28 days later. Images of AXL (red), MARCO (white), LYVE-1 (green) and DAPI (nuclei, blue) were acquired ( C ). Computerized quantification of the staining densities ( D ) and spatial distribution analysis ( E , F ) were performed in murine Sham and Td LNs. D. Data are means ± SEM for LYVE-1 and MARCO graphs and medians with interquartile ranges for AXL graph. Statistical tests: Unpaired t test (LYVE-1 and MARCO graphs) and Mann–Whitney U test (AXL graph). * p < 0.05, ** p < 0.01. E. Data are staining area medians according to the distance from the LN border (d = 0). F. Data are p-values of staining area differences between Td and Sham LNs for each distance. Below the dotted line, p is lower than 0.05 and considered as significant. Statistical test: Mann–Whitney U test for each distance. n ≥ 7 for each group. G. Images of Prox1 RNAscope hybridization (lymphatic vessel, green) and AXL immunostaining (red) in Sham and Td LNs

Article Snippet: Cryosections were incubated at room temperature for 1 h with AXL and MARCO (Bio-Rad Cat# MCA1849T, RRID: AB_2140591, 1/200) antibodies.

Techniques: In Vivo, Staining, MANN-WHITNEY, RNAscope, Hybridization, Immunostaining

Journal: European journal of immunology

Article Title: Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages.

doi: 10.1002/eji.202350771

Figure Lengend Snippet: Figure 2. (A, B) Wildtype and Marco−/−macrophages were stimulated with 10 ng/mL LPS overnight, then infected with anti-GXM 18B7 antibody opsonised C. neoformans. After 2 h infection, images were acquired every 5 min for 16 h. (B) Wildtype: n = 27 vomocytosis events out of 220 infected macrophages; Marco−/−: n = 153 vomocytosis events out of 166 infected macrophages. (C, D) LPS-stimulated macrophages were infected with a yeast- locked TetO-NRG1 C. albicans strain. Images were acquired every 5 min for 6 h. The phagocytic index (%) represents the percentage of macrophages that phagocytosed one or more fungal cells. At least 200 macrophages were observed per condition. Vomocytosis (%) is the percentage of infected macrophages that experienced at least one expulsion event. (D) Wildtype: n = 4 vomocytosis events out of 112 infected macrophages; Marco−/−: n = 36 vomocytosis events out of 101 infected macrophages. (E) Representative image showing vomocytosis of C. albicans from Marco−/−MPI cells. Time is presented in hh:mm:ss; red arrows follow the course of a vomocytosis event; scale bar = 10 μm. (F, G) Wildtype macrophages were stim- ulated overnight with 10 ng/mL LPS. The following day, cells were pre-treated with polyguanylic acid (polyG) or (H, I) anti-MARCO ED31 mono- clonal antibody (mAb) for 30 min then infected with non-opsonised C. neoformans still in the presence of polyG or anti-MARCO mAb. Images were acquired every 5 min for 16 h. Phagocytosis was quantified as the total number of internalised fungi per 100 macrophages at the beginning of the

Article Snippet: Meanwhile, to follow infection from uptake through to vomocytosis, timelapse imaging began immediately following macrophage incubation with C. neoformans at an MOI 0.5:1.Where applicable, macrophages were pre-treated with 400 μg/mL polyG (Sigma-Aldrich; cat#: P4404), rat antimouse MARCO ED31 clone monoclonal antibody (BioRad; Cat#: MCA1849), or anti-rat IgG1 isotype control (Invitrogen; cat#: 14430182) for 30 min at 37°C prior to 2 h infected with nonopsonised C. neoformans.

Techniques: Infection

Journal: European journal of immunology

Article Title: Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages.

doi: 10.1002/eji.202350771

Figure Lengend Snippet: Figure 2. (A, B) Wildtype and Marco−/−macrophages were stimulated with 10 ng/mL LPS overnight, then infected with anti-GXM 18B7 antibody opsonised C. neoformans. After 2 h infection, images were acquired every 5 min for 16 h. (B) Wildtype: n = 27 vomocytosis events out of 220 infected macrophages; Marco−/−: n = 153 vomocytosis events out of 166 infected macrophages. (C, D) LPS-stimulated macrophages were infected with a yeast- locked TetO-NRG1 C. albicans strain. Images were acquired every 5 min for 6 h. The phagocytic index (%) represents the percentage of macrophages that phagocytosed one or more fungal cells. At least 200 macrophages were observed per condition. Vomocytosis (%) is the percentage of infected macrophages that experienced at least one expulsion event. (D) Wildtype: n = 4 vomocytosis events out of 112 infected macrophages; Marco−/−: n = 36 vomocytosis events out of 101 infected macrophages. (E) Representative image showing vomocytosis of C. albicans from Marco−/−MPI cells. Time is presented in hh:mm:ss; red arrows follow the course of a vomocytosis event; scale bar = 10 μm. (F, G) Wildtype macrophages were stim- ulated overnight with 10 ng/mL LPS. The following day, cells were pre-treated with polyguanylic acid (polyG) or (H, I) anti-MARCO ED31 mono- clonal antibody (mAb) for 30 min then infected with non-opsonised C. neoformans still in the presence of polyG or anti-MARCO mAb. Images were acquired every 5 min for 16 h. Phagocytosis was quantified as the total number of internalised fungi per 100 macrophages at the beginning of the

Article Snippet: Meanwhile, to follow infection from uptake through to vomocytosis, timelapse imaging began immediately following macrophage incubation with C. neoformans at an MOI 0.5:1.Where applicable, macrophages were pre-treated with 400 μg/mL polyG (Sigma-Aldrich; cat#: P4404), rat antimouse MARCO ED31 clone monoclonal antibody (BioRad; Cat#: MCA1849), or anti-rat IgG1 isotype control (Invitrogen; cat#: 14430182) for 30 min at 37°C prior to 2 h infected with nonopsonised C. neoformans.

Techniques: Infection