Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Secretomic analysis of metastasis-related differentially expressed proteins in oral cancer cells. (a) After sample preparation of each cell condition, protein samples were lysed and arranged for a triplicate 2D-DIGE experiment. The black and white images are shown as the protein profiles of the OC3 and OC3-IV2 cells. Differentially expressed proteins are indicated as red or green spots in the overlaid pseudo-colored image. (b) Numerous protein spots annotated with yellow circles represent the differentially expressed proteins with greater than 2-fold or lesser than −2-fold differences using the DeCyder ™ software. (c) Upregulated LGALS1 in OC3-IV2 cells was revealed according to the 2D image, 3D image, and statistical data from the DeCyder ™ analysis. (d) There is no specific loading control for secreted proteins. The whole secreted proteins were used as protein loading control. (e) Top: validation by immunoblotting analysis between OC3 and OC3-IV2 cells. Bottom: relative LGALS1 expression was quantified and normalized with respect to the loading control. Data derived from three independent experiments are presented as mean ± SEM. **, p < 0.01 when compared to OC3. 2D-DIGE, two-dimensional difference gel electrophoresis; LGALS1, lectin, galactoside-binding, soluble, 1; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Sample Prep, Software, Control, Biomarker Discovery, Western Blot, Expressing, Derivative Assay, Nucleic Acid Electrophoresis, Binding Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: LGALS1 protein expression in tissue specimens of oral cancer. (a) Clinical tissue samples of oral cancer on the tissue microarray comprises of normal/adjacent normal tissue, malignant tumor, and lymph node metastatic tissue. LGALS1 expression was examined by immunochemical staining. From weak to strong, LGALS1 expression during different stages of the clinical samples (top, scale bar: 500 μm). The bottom images represent the magnification of the tissues (scale bar: 100 μm). (b) The LGALS1 staining intensity in normal/adjacent normal tissue ( n = 10), malignant tumor ( n = 42), and lymph node metastasis ( n = 4) was assessed using ImageJ. Tukey’s post-hoc test for statistical analysis of LGALS1 expression was performed to clarify the role of LGALS1 in clinical samples. Data are represented as mean ± SEM. *, p < 0.05 when compared to the malignant tumor group and **, p < 0.01 when compared to the normal/adjacent normal tissue group. LGALS1, lectin, galactoside-binding, soluble, 1; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Expressing, Microarray, Staining, Binding Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Effect of LGALS1 knockdown on cell proliferation and cell cycle regulation in oral cancer. (a, b) OC3, OC3-IV2, C9, and C9-IV2 cells were transfected with 40 nM of siCtrl or siLGALS1, and the secreted protein samples of each cell line were collected and concentrated from the conditioned medium. The knockdown efficiency of secreted LGALS1 was monitored via immunoblotting analysis. Coomassie blue images demonstrate the protein loading control. (c, d) Cell proliferation was observed from Day 0 to Day 3 using the MTT assay. The values of Day 1, Day 2, and Day 3 were normalized to Day 0. Data are represented as mean ± SEM. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 when compared to siCtrl. (e, f) Representative plots of cell cycle analysis using a flow cytometer. siCtrl- or siLGALS1-transfected OC3 and OC3-IV2 cells were stained with PI to analyze DNA content. The percentage of cells in the G 0 /G 1 , S, and G 2 /M phases were displayed on the FL2-A fluorescent channel of the flow cytometer. The percentage of total cells at different stages were statistically analyzed on the right histogram. Data derived from three independent experiments are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 when compared to siCtrl. (g) Cell cycle regulatory proteins were differentially expressed in response to LGALS1 knockdown. Immunoblotting analysis of cyclin D2, cdk4, cyclin E, cyclin A2, cdk2, and p27 proteins in OC3 and OC3-IV2 cells transfected with control or LGALS1 siRNA was carried out. Immunoblotting results of the indicated proteins were quantified by ImageJ and normalized to GAPDH. The protein intensity values were relative to the siCtrl-transfected OC3 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LGALS1, lectin, galactoside-binding, soluble, 1; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Knockdown, Transfection, Western Blot, Control, MTT Assay, Cell Cycle Assay, Flow Cytometry, Staining, Derivative Assay, Binding Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Effect of LGALS1 knockdown on wound healing ability and oral cancer cell migration. (a, b) The wound areas were generated by scratching with 10 μl pipette tips. The wound healing of oral cancer cells was observed and photographed at 0 h, 8 h, 12 h, and 24 h using an optical microscope (scale bar: 100 μm). Bottom: wound areas at the indicated times were calculated and quantified using AxioVision Rel. 4.8 software. The values for the relative wound area were normalized to the values at 0 h. Data are represented as mean ± SEM. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 when compared to siCtrl-transfected oral cancer cells. (c, d) Transwell migration assay of oral cancer cells transfected with siCtrl or siLGALS1. Representative images are displayed at a 40 × magnification using an optical microscope. The migrated cells were stained with crystal violet and then quantified in solution using an ELISA reader. The values for the relative migration ability were normalized to the siCtrl of each oral cancer cell line. Data derived from three independent experiments are presented as mean ± SEM. **, p < 0.01 and ***, p < 0.001 when compared to siCtrl-transfected cells. ELISA, enzyme-linked immunosorbent assay; LGALS1, lectin, galactoside-binding, soluble, 1; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Knockdown, Migration, Generated, Transferring, Microscopy, Software, Transfection, Transwell Migration Assay, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Binding Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Effect of LGALS1 knockdown on oral cancer cell invasion and in vivo metastasis ability. (a, b) Control or LGALS1 siRNA-transfected oral cancer cells were subjected to the matrigel invasion assay. The quantified values of the relative invasion ability were normalized to the siCtrl of the cell lines. Data derived from three independent experiments are presented as mean ± SEM. *, p < 0.05 and **, p < 0.01 when compared to siCtrl-transfected cells. (c) In vivo lung metastasis of siCtrl- or siLGALS1-transfected C9-IV2 cells via the tail vein injection. Lung tissues were examined by paraffin section and H&E staining. Top: H&E staining images (scale bar: 1 mm). Bottom: The zoomed images of the indicated areas (scale bar: 100 μm). Arrowhead: tumor sites of lung metastases. (d) Lung metastasis of the injected cells were determined by specific primers for hGAPDH using qPCR. Relative hGAPDH expression of the siCtrl group ( n = 7 mice) and siLGALS1 group ( n = 7 mice) were normalized to mouse actin mRNA. Data is represented as mean ± SEM. *, p < 0.05 when compared to the siCtrl group. H&E, hematoxylin and eosin; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; LGALS1, lectin, galactoside-binding, soluble, 1; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Knockdown, In Vivo, Control, Transfection, Invasion Assay, Derivative Assay, Injection, Paraffin Section, Staining, Expressing, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Role of LGALS1 on MMPs expression, MAPK signaling pathway, and EMT status in oral cancer cells. (a, b) Differential expression of the MMP-9 and MMP-3 proteins from the conditioned medium. The Coomassie blue staining images served as protein loading controls for normalization. The protein expression values were relative to siCtrl-transfected OC3 or C9 cells. (c, d) Immunoblotting analysis of phosphorylated p38 MAPK (p-p38 MAPK) and ERK (p-ERK) normalized to whole proteins (p38 MAPK or ERK). The quantified values were relative to OC3 or C9 cells transfected with control siRNA and statistically analyzed on the right histogram. Data derived from three independent experiments are presented as mean ± SEM. *, p < 0.05 and **, p < 0.01 when compared to siCtrl-transfected cells. (e, f) Representative images of the EMT-regulating transcription factors (ZEB2, SNAI1, TWIST) and EMT markers (E-cadherin, N-cadherin, and vimentin) normalized to GAPDH in siCtrl- or siLGALS1-transfected oral cancer cells. The protein expression values were quantified in relation to the siCtrl-transfected OC3 or C9 cells. EMT, epithelial-mesenchymal transition; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LGALS1, lectin, galactoside-binding, soluble, 1; p38 MAPK, p38 mitogen-activated protein kinase; MMP, matrix metalloproteinase; n.s., nonsignificant; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Expressing, Quantitative Proteomics, Staining, Transfection, Western Blot, Control, Derivative Assay, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Therapeutic Advances in Medical Oncology

Article Title: Upregulation of LGALS1 is associated with oral cancer metastasis

doi: 10.1177/1758835918794622

Figure Lengend Snippet: Effect of elevated LGALS1 and p38 MAPK knockdown on oral cancer cell migration. (a) Expression of p-p38 MAPK following treatment with rhLGALS1 (200 ng/ml) was examined by immunoblotting. The expression of p-p38 MAPK was normalized to that of whole p38 MAPK. The quantified values were relative to those in untreated parental OC3 or C9 cells. (b) Untreated or rhLGALS1 (500 ng/ml)-treated oral cancer cells were subjected to the transwell migration assay. The quantified values of the relative migration ability were normalized to the untreated parental cells. Data derived from three independent experiments are presented as mean ± SEM. **, p < 0.01 and ***, p < 0.001 when compared to untreated OC3 or C9 cells. (c) OC3-IV2 and C9-IV2 cells were transfected with 40 nM of siCtrl or sip38 MAPK, and the knockdown efficiency of p38 MAPK was monitored via immunoblotting analysis. (d) Transwell migration assay of highly invasive oral cancer cells transfected with siCtrl or sip38 MAPK. Data derived from three independent experiments are presented as mean ± SEM. ***, p < 0.001 when compared to siCtrl-transfected cells. (e) Immunoblotting analysis of the MMP-9 proteins from the conditioned medium. The Coomassie blue staining images served as protein loading controls for normalization. The protein expression values were relative to siCtrl-transfected OC3-IV2 or C9-IV2 cells. (f) A hypothetical model detailing the role of elevated LGALS1 in metastatic oral cancer cells. Overexpressed LGALS1 in highly invasive oral cancer cells interacts with integrins in the extracellular space and subsequently induces the phosphorylation of p38 MAPK. The activation of the p38 MAPK pathway accelerates MMP-9 expression, which facilitates the secretion and upregulation of ZEB2, SNAI1, and TWIST in order to induce EMT. Thus, increased LGALS1 activates p38 MAPK and augments the expression of secreted MMP-9 as well as the EMT-regulating transcription factors to facilitate migration and invasion in oral cancer cells. EMT, epithelial-mesenchymal transition; rhLGALS1, recombinant human lectin, galactoside-binding, soluble, 1; p38 MAPK, p38 mitogen-activated protein kinase; MMP, matrix metalloproteinase; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: For immunochemical staining of LGALS1 within the tissue array slide, specimens were incubated with anti-LGALS1 antibody (Genetex).

Techniques: Knockdown, Migration, Expressing, Western Blot, Transwell Migration Assay, Derivative Assay, Transfection, Staining, Phospho-proteomics, Activation Assay, Recombinant, Binding Assay, Real-time Polymerase Chain Reaction

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: bioRxiv

Article Title: Aged blood inhibits hippocampal neurogenesis and activates microglia through VCAM1 at the blood-brain barrier

doi: 10.1101/242198

Figure Lengend Snippet: Related to (a) Schematic of flow sorting of CD31+CD45-BECs from mouse cortex and hippocampi. Each isolated RNA sample is a pool of BECs from 2 mouse brains. (b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia. CD31+Cd11b-CD45-cells were defined as the BEC population. (c) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of CNS cell-type specific markers. (d) FPKM values of leukocyte binding adhesion molecules including Vcam1 . (e) FPKM values of the gene transcripts in murine young and aged CD31+BECs of human plasma proteins that change with age (see for list of human plasma proteins expressed in murine BECs) (f) C57BL6 mice were injected with anti-VCAM1-DL488 or IgG isotype control (r.o.) 2 hours before perfusion to label BECs in vivo prior to brain dissociation, staining and FACS. (g) Gating and histogram plots of CD31+VCAM1+ cells sorted from the hippocampi of LPS stimulated aged (19-month old) wildtype mice injected with fluorescently tagged anti-VCAM1 mAb or IgG isotype (h) Quantification of CD31+VCAM1+cells isolated from healthy cortex (n=5 single mice/age group). **p<0.002 (i) Quantification of CD31+VCAM1+cells isolated from 4 pooled hippocampi (technical replicates shown). **p<0.005 (j) Flow gating and histogram plots of pooled (n=4 mice/ age group), young or aged hippocampi isolated from healthy mice also injected with anti-VCAM1 mAb. (k) sVCAM1 ELISA in plasma from young isochronic or heterochronic parabionts following 5 weeks of parabiosis. n=11 mice/group pooled from two independent experiments. **p<0.004, Student’s t-test . All error bars indicate SEM. (l) Confocal images in the DG of VCAM1, lectin, and Aqp4 of young isochronic or heterochronic parabionts 5 weeks after surgery. Hoechst labels cell nuclei. Scale bar = 100 μm.

Article Snippet: Primary: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326), Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C10637), goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc17320), mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI), rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569), DyLight 488 Lectin (1:200, Vector, DL-1174), rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218), rat monoclonal anti-CD68 (1:600, Serotec, MCA1957), goat polyclonal anti-Iba1 (1:250, ProteinTech, 10904-1-AP), goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc8066), mouse anti-human-VCAM1 antibody (BBA5, Novus Biologicals), monoclonal mouse anti-human IgG antibody (MAB002, R&D Systems); rat monoclonal anti-VCAM-1 (clone M/K-2.7, Bioxell, BE0027); Rat IgG 1 Isotype antibody (Clone HRPN, Bioxell, BE0088), VE-Cadherin (sc-6458, Santa Cruz Biotechnology), FC blocking antibody (553142, BD Pharmigen), rat anti-CD31 antibody (CD31-APC) (551262, BD Pharmigen), Dylight 488 Conjugation Kit (53024, Thermo Scientific), Anti-Mouse CD45 PerCP-Cyanine5.5 (1:1000, eBioscience, 45-0451-80), PerCP/Cy5.5 anti-mouse CD11a/CD18 (LFA-1) (1:100, Biolegend, 141007), Anti-Mouse CD11b PerCP-Cyanine5.5 (1:100, eBioscience, 45-0112-80), Anti-Mouse TER-119 PerCP-Cyanine5.5 (1:100, eBioscience, 45-5921-80), CD13 Antibody (ER-BMDM1) APC (1:50, NOVUS Biologicals, NB100-64843), Anti-ACSA-2-PE mouse (clone: IH3-18A3) (1:100, Miltenyi Biotec Inc., 130-102-365), Anti-Mouse CD31 (PECAM-1) PE-Cyanine7 (1:100, eBioscience, 25-0311-81), Anti-mouse MECA-99 antibody was a gift of the Butcher lab and labeled with fluorophores using DyLight™ Antibody Labeling Kit (DyLight™488, Thermo Scientific, 53025), CD31-APC (1:100, BD 551262), CD45-FITC (1:100, BD Pharmingen Clone 30-F11 553080), and Cd11b-BV421 (1:100, Biolegend Clone M1/70 101236) Secondary: Alexa Fluor® 488 donkey anti-goat IgG (1:250, Thermo/Life Technologies, A-11055), Alexa Fluor® 488 donkey anti-rat IgG (Invitrogen/Life Technologies, A21208), Alexa Fluor® 555 donkey anti-mouse IgG (1:250, Invitrogen, A31570), Alexa Fluor® 555 donkey anti-goat IgG (1:250, Invitrogen, A21432), Cy3 AffiniPure donkey anti-rat IgG (1:250, Jackson Immunoresearch, 712-165-153), Alexa Fluor® 647 donkey anti-mouse IgG (1:250, Invitrogen, A31571), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor® 647 donkey anti-rabbit IgG (1:250, Life Technologies, A-31573), Alexa Fluor® 647 donkey anti-goat IgG (1:250, Invitrogen, A-21447), Alexa Fluor 488 Azide (A10266, Life Technologies); Alexa Fluor 647 Azide (A10277, Life Technologies); Hoechst 33342 (1:2000, Sigma, 14533-100MG)

Techniques: Isolation, Binding Assay, Injection, In Vivo, Staining, Enzyme-linked Immunosorbent Assay