Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and group quantitation for circulating Ly6C low CD169 + Tim4 + macrophages in sham-operated and MI wild type (WT) C57BL/6 mice 1 d post-MI; n=4/group, statistics: unpaired t test. B. Left , Flow histograms demonstrating surface expression of CCR3 and CCR4 in Ly6C low CD169 + Tim4 + macrophages from the same groups; statistics: unpaired t test, NTM, normalized to mode, y-axis represents cell counts. Right , chemokine gene expression by RT-PCR (normalized to 18s) in the myocardial border zone (BZ) 1 d after MI or sham operation; n=4-5/group, statistics: unpaired t test. *p<0.05, **p<0.01, ***p<0.001 versus sham. C . Left , Circulating Ly6C low CD169 + Tim4 + cell frequency prior to and 1 d after MI in Spx mice; n=9, statistics: paired t test. NS, not significant. D. FACS density plots, histograms, and quantitation of cardiac Ly6C low CD169 + Tim4 + macrophages in WT and Spx mice 1 d post-MI or sham operation; n=4-7/group, statistics: unpaired t test. E . Immunostains and FACS dot plots of splenic CD169 + MMMs (red) 24 h after MI or sham operation, and quantitation of MMM frequency by FACS and spleen weight (Wt); n=5-7/group, statistics: unpaired t test. TL, tibia length. F . Left , FACS dot plots and histograms, and corresponding quantitation, of blood and heart Ly6C low CD169 + Tim4 + Bioparticle + cells from sham and MI mice given 10 mg/kg Texas Red-conjugated bioparticles i.v. 3 h before sacrifice; n=3-4/group, statistics: unpaired t test for blood and non-parametric Mann-Whitney U test for heart (non-normal distribution). Right, quantitation of splenic BioParticle + MMMs in the same experimental mouse groups; statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left, FACS pseudocolor plots of cardiac CD169 + Tim4 + macrophages and separation based on LYVE1 surface expression in naïve and 1 d post-MI mice. Right , quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in hearts from naïve and 1 d post-MI mice. n=4-6/group; statistics: unpaired t test. B . Heat map of 462 significant (p adjusted<0.05) DEGs by RNAseq analysis in sorted LYVE1 hi and LYVE1 low CD169 + Tim4 + cardiac macrophages 1 d post-MI. C. PCA plots using the top 500 DEGs after rlog transformation of RNAseq data from these macrophages sorted from the indicated sites in naïve and 1 d post-MI mice. D. Left , Flow histograms depicting LYVE1 surface expression on cardiac CD169 + Tim4 + macrophages (green) and total Autofluorescence + CD64 + MHCII + macrophages (brown) in WT and Spx mice, 1 d post-MI. Right , FACS quantitation of LYVE1 hi and LYVE1 low CD169 + Tim4 + macrophages in the hearts of WT and Spx mice, 1 d post-MI; n=5-6/group. Statistics: unpaired t test. E. Representative confocal micrograph of border zone (BZ) myocardium immunostained for CD169 (red) and Tim4 (green) 1 d post-MI in WT and Spx mice; nuclear staining with DAPI (blue). Scale bar, 200 μm. Inset shows magnified images of CD169 and Tim4 staining. Yellow arrows indicate double positive cells; scale bar 10 μm.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Expressing, Quantitation Assay, Transformation Assay, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Protocol for LXRα agonist T0901317 treatment (40 mg/kg i.p.) from 1 d prior to 5 d post-MI, with 10 d post-MI follow-up, in WT and Spx mice. B . FACS contour plots and quantitation of cardiac CD169 + Tim4 + macrophages 1 d post-MI and blood CD169 + Tim4 + macrophages 10 d post-MI in untreated and T0901317-treated WT and Spx mice. N=5-6/group, statistics: one-way ANOVA, Bonferroni post-test. C . Kaplan-Meier survival curves post-MI in untreated and T0901317-treated WT and Spx mice. Statistical comparisons by log-rank test, group sizes as indicated. D . Representative end-diastolic long-axis 2-dimensional echocardiograms and group data for LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) in the same mouse groups at 10 d post-MI; n=5-10/group, statistics: one-way ANOVA, Bonferroni post-test. E . Top , Kaplan-Meier survival curves over 8 w post-MI in WT mice treated with either vehicle or T0901317 from 1 d before MI to 5 d post-MI (statistical comparison by log-rank test, n=12-17/group as indicated) and group data for LVEF, LVEDV, LVESV, and normalized heart and lung weight at 8 w post-MI; n=8-9/group for echocardiography, n=4-7/group for gravimetry. Statistical comparisons: unpaired t test. HF, heart failure; TL, tibia length; NS, not significant. Bottom Left , Representative Masson’s trichrome staining of LV short-axis sections (2x magnification) and infarct border zone (BZ, scale bar 500 μm), along with quantitation of cardiac fibrosis (BZ and remote zone [RZ], blue staining) in vehicle- and T0901317-treated WT HF mice. Also shown are confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the RZ of hearts from vehicle-and T0901317-treated HF mice (8 w post-MI) and quantitation of iNOS + CD206 + and iNOS − CD206 + macrophages (Mφ) per mm 2 . Double positive (CD206 + iNOS + ) cells appear yellow (arrows). DAPI (blue) was used for nuclear staining. N=4-8/group, statistics: unpaired t test. Bottom Right , Representative FACS contour plots to identify Ly6C hi monocytes in vehicle- and T0901317-treated WT HF mice (8 w post-MI), and corresponding quantitation. N=5-10/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Quantitation Assay, Comparison, Staining

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Control, Quantitation Assay

Journal: Discover Oncology

Article Title: SIGLEC1 has the potential to be an immune-related prognostic indicator in colon adenocarcinoma: a study based on transcriptomic data and Mendelian randomization analysis

doi: 10.1007/s12672-025-02093-2

Figure Lengend Snippet: Protein–protein interaction network and univariate COX. A Gene networks from the module gene based on the WGCNA method. B Univariate Cox regression analysis, listing to prognosis. C , D Overall survival analysis of the CYHR1 and SIGLEC1 genes in colon cancer based on the Kaplan–Meier plotter

Article Snippet: Immunohistochemical staining was conducted using primary anti-SIGLEC1 antibody (1:200; 55427-1-AP, proteintech, China).

Techniques:

Journal: Discover Oncology

Article Title: SIGLEC1 has the potential to be an immune-related prognostic indicator in colon adenocarcinoma: a study based on transcriptomic data and Mendelian randomization analysis

doi: 10.1007/s12672-025-02093-2

Figure Lengend Snippet: GSEA delineates high SIGLEC1 expression and low expression. A HALLMARK collection enriched in the high SIGLEC1 expression sample. B HALLMARK collection enriched in the low SIGLEC1 expression sample. C Enriched immunologic gene sets in C7 collection of the high SIGLEC1 expression group. D Enriched immunologic gene sets in C7 collection of the low SIGLEC1 expression group

Article Snippet: Immunohistochemical staining was conducted using primary anti-SIGLEC1 antibody (1:200; 55427-1-AP, proteintech, China).

Techniques: Expressing

Journal: Discover Oncology

Article Title: SIGLEC1 has the potential to be an immune-related prognostic indicator in colon adenocarcinoma: a study based on transcriptomic data and Mendelian randomization analysis

doi: 10.1007/s12672-025-02093-2

Figure Lengend Snippet: Significant correlation between the expression of SIGLEC1 and the proportion of tumor-infiltrating immune cells. A Scatter plot showing the correlation between 10 types of tumor-infiltrating immune cell ratio with the SIGLEC1 expression (all p < 0.05). B Mendelian randomization analysis. C Correlation between the immune checkpoint genes between SIGLEC1 expression

Article Snippet: Immunohistochemical staining was conducted using primary anti-SIGLEC1 antibody (1:200; 55427-1-AP, proteintech, China).

Techniques: Expressing

Journal: Discover Oncology

Article Title: SIGLEC1 has the potential to be an immune-related prognostic indicator in colon adenocarcinoma: a study based on transcriptomic data and Mendelian randomization analysis

doi: 10.1007/s12672-025-02093-2

Figure Lengend Snippet: Patients’ characteristics and clinicopathological parameters of the study group

Article Snippet: Immunohistochemical staining was conducted using primary anti-SIGLEC1 antibody (1:200; 55427-1-AP, proteintech, China).

Techniques: Expressing

Journal: Discover Oncology

Article Title: SIGLEC1 has the potential to be an immune-related prognostic indicator in colon adenocarcinoma: a study based on transcriptomic data and Mendelian randomization analysis

doi: 10.1007/s12672-025-02093-2

Figure Lengend Snippet: Clinical COAD samples validate SIGLEC1 signature. A The immunohistochemical (IHC) staining on clinical sample tissue microarrays indicates high and low expression of SIGLEC1. B Kaplan–Meier analysis of SIGLEC1 expression levels

Article Snippet: Immunohistochemical staining was conducted using primary anti-SIGLEC1 antibody (1:200; 55427-1-AP, proteintech, China).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Journal of Bioactive and Compatible Polymers

Article Title: Potential of mannan or dextrin nanogels as vaccine carrier/adjuvant systems

doi: 10.1177/0883911516631354

Figure Lengend Snippet: Figure 3. Fluorescence observation of lymph node cryosections stained with anti-CD169 (red) to identify macrophages and with streptavidin dylight 488 to identify biotin-labeled nanogels (green) after footpad injection of nanogels: (a) popliteal node as negative control (without nanogel injection); (b) popliteal node 24 h after footpad injection of mannan nanogels, and (c) popliteal node 24 h after footpad injection of dextrin nanogels,. Nuclei are stained in blue. All images are representative of two independent experiments. Scale bars: 300 µm.

Article Snippet: The primary antibody mouse anti-rat CD169 (clone ED3) was purchased from Serotec (USA) and 4′,6-diamidino-2-phenylindole (DAPI) containing mounting media (Vectashield H200; Vector, USA).

Techniques: Fluorescence, Staining, Labeling, Injection, Negative Control