primary antibodies against tim 4  (Proteintech)

Journal: The Journal of Experimental Medicine

Article Title: XPR1 regulates fetal liver macrophage development, identity, and pyrenocyte clearance

doi: 10.1084/jem.20241587

Figure Lengend Snippet: FL macrophage development requires Xpr1 expression in hematopoietic cells. (A) Representative flow cytometry plots and total cell numbers (mean ± SD) of FL macrophages (Mac) extracted from E16.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl embryos (pre-gated on CD45 + Lin − [CD19 − CD3 − B220 − Ter119 − CD49b − CD90.2 − ] cells). Data show n = 4–8 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (B) Graphs showing embryo and liver weights of E18.5 Xpr1 fl/+ , Xpr1 fl/fl , Vav1 iCre Xpr1 fl/+ , and Vav1 iCre Xpr1 fl/fl embryos. Data show n = 5–9 per group and were pooled from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. (C) Representative flow cytometry plots and total cell numbers (mean ± SD) of liver Ly6C hi monocytes (Mo) extracted from E16.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl embryos (pre-gated on CD45 + Lin − F4/80 − CD117 lo-int CD48 − cells). Data show n = 4–8 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (D) UMAP plots of myeloid cells extracted from livers of E16.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl embryos (pre-gated on CD45 + Lin − CD64 + F4/80 + MHCII − Ly6G − cells) and analyzed by flow cytometry. Data were generated from n = 2–4 samples per group and are representative of at least four independent experiments. (E) Graphs showing frequencies of cell clusters defined among CD45 + cells ( D ). Data show n = 2 per group and are representative of at least four independent experiments. (F) Heatmap of cell surface marker expression on cell clusters defined in D. Data were transformed and percentile normalized. (G) Immunohistochemistry of E16.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl livers. DAPI (blue), CD64 (red), and F4/80 (green). Insets: Magnifications of the outlined regions showing expression of all markers, CD64/DAPI, and F4/80/DAPI (from left to right). Images are representative of at least five embryos per group. Scale bar: 40 µm. (H) Immunohistochemistry of E15.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl livers, stained for Hoechst (blue), IBA1 (cyan), and TIM4 (white). Insets: Single stainings. Images are representative of three embryos per group. Scale bar: 100 µm in overview, 50 µm in inset. **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Article Snippet: Primary antibodies used were F4/80 (clone BM8, 123101; BioLegend; clone CI:A3-1, MCA497A488; Bio-Rad), CD64 (clone AT152-9, MCA5997; Bio-Rad), IBA1 (ABIN2857032; Antibodies Online), TIM4 (AF2826; R&D Systems), CD31 (AF3628; R&D Systems), and CD206 (GTX42265; GeneTex).

Techniques: Expressing, Flow Cytometry, Comparison, Generated, Marker, Transformation Assay, Immunohistochemistry, Staining

Journal: The Journal of Experimental Medicine

Article Title: XPR1 regulates fetal liver macrophage development, identity, and pyrenocyte clearance

doi: 10.1084/jem.20241587

Figure Lengend Snippet: Phenotypic and functional changes in Mrc1 Cre Xpr1 fl/fl animals. (A) Violin plots showing the expression of Mrc1 in the indicated clusters of E15.5 FL cells. Data refer to scRNA-seq data in . (B) Immunohistochemistry of E15.5 Xpr1 fl/fl control liver stained for IBA1 (cyan), CD206 (magenta), and CD31 (white). Insets: Single stainings shown of the outlined region in the overview image. Scale bar: 100 µm, inset 50 µm. Filled arrowheads show CD206 + macrophages, open arrowheads show CD206 + endothelial cells. (C) Representative flow cytometry plot of CD206 expression in liver macrophages extracted from E15.5 Xpr1 fl/fl embryos (pre-gated on CD45 + CD31 − Ly6G − F4/80 + CD11b + TER119 − cells). (D) Representative flow cytometry plots of Tim4 expression in liver macrophages extracted from E15.5 Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl embryos (pre-gated on CD45 + Ly6G − F4/80 + cells). Data are representative of at least n = 5 per group. (E) Representative flow cytometry plots and graph showing the percentage (normalized to control) of pHrodo Red + FL macrophages (gated on CD45 + F4/80 + CD11b int cells) from Mrc1 Cre Xpr1 fl/fl and Xpr1 fl/fl FLs at E15.5, exposed in vitro to pHrodo Red Zymosan bioparticles for 90 min. Data shown are n = 3–6 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (F) Heatmap of all 6,208 DEGs in the RNA-seq data of Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl FL macrophages from . Genes indicated are the core KC-genes and KC-associated transcription factors identified in . (G) Gene ontology analysis for biological processes of the DEGs in the RNA-seq data of Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl FL macrophages from . (H) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of Tim4 expression on KCs of adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice (pre-gated on CD45 + Ly6G − F4/80 + cells). Data shown are n = 3–5 per group and are pooled from two independent experiments. Statistical analyses were performed using the Student’s t test. (I) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of KCs of adult Xpr1 fl/fl and Siglec1 Cre Xpr1 fl/fl mice (pre-gated on CD45 + Ly6G − F4/80 + cells). Data shown are n = 3 per group. Statistical analyses were performed using the Student’s t test. ( J–M ) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of (J) heart macrophages (Heart Macs) (pre-gated on CD45 + Ly6G − cells), (K) Langerhans cells (LCs) (pre-gated on CD45 + Ly6G − TCRβ − SiglecF − Langerin + cells), (L) kidney macrophages (Kidney Macs) (pre-gated on CD45 + Ly6G − CD3 − CD19 − B220 − CD49b − CD90 − cells), and (M) alveolar macrophages (AMs) (pre-gated on CD45 + Ly6G − CD3 − CD19 − B220 − CD49b − CD90 − cells) of adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice. Data shown are n = 5 per group and are pooled from two independent experiments. Statistical analyses were performed using the Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Article Snippet: Primary antibodies used were F4/80 (clone BM8, 123101; BioLegend; clone CI:A3-1, MCA497A488; Bio-Rad), CD64 (clone AT152-9, MCA5997; Bio-Rad), IBA1 (ABIN2857032; Antibodies Online), TIM4 (AF2826; R&D Systems), CD31 (AF3628; R&D Systems), and CD206 (GTX42265; GeneTex).

Techniques: Functional Assay, Expressing, Immunohistochemistry, Control, Staining, Flow Cytometry, In Vitro, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: XPR1 regulates fetal liver macrophage development, identity, and pyrenocyte clearance

doi: 10.1084/jem.20241587

Figure Lengend Snippet: XPR1 regulates iron-recycling macrophages in the spleen and BM. (A) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of splenic macrophages from E18.5 control ( Xpr1 fl/fl and Xpr1 fl/+ ) and Vav1 iCre Xpr1 fl/fl embryos (pre-gated on CD45 + Lin − Ly6C − Ly6G − SiglecF − CD64 + cells). n = 3–10 per group, pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (B) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of splenic Ly6C hi monocytes from E18.5 Xpr1 fl/fl and Vav1 iCre Xpr1 fl/fl embryos (pre-gated on CD45 + Lin − Mac − SiglecF − cells). Data show n = 3–10 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (C) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of splenic red pulp macrophages (RPMs) from adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice (pre-gated on CD45 + Lin − Ly6G − SiglecF − CD90 − MHCII − cells). Data show n = 3–5 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (D) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of splenic Ly6C hi monocytes from adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice (pre-gated on CD45 + Lin − Ly6G − SiglecF − CD90 − B220 − CX3CR1 + CD64 + cells). Data show n = 3–5 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (E) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of F4/80 + MerTK + BM macrophages (top) (pre-gated on CD45 + Lin − Ly6C − Ly6G − ) and among these, CD163 + TIM4 + macrophages (bottom) from adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice. Data show n = 4–6 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (F) Representative flow cytometry plots, frequencies, and numbers (mean ± SD) of BM monocytes (CD115 + CX3CR1 + ) from adult Xpr1 fl/fl and Mrc1 Cre Xpr1 fl/fl mice (pre-gated on CD45 + Lin − Ly6G − Ly6C + SiglecF − ). Data show n = 4–6 per group and are pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. ***P < 0.001 and ****P < 0.0001. ns, not significant.

Article Snippet: Primary antibodies used were F4/80 (clone BM8, 123101; BioLegend; clone CI:A3-1, MCA497A488; Bio-Rad), CD64 (clone AT152-9, MCA5997; Bio-Rad), IBA1 (ABIN2857032; Antibodies Online), TIM4 (AF2826; R&D Systems), CD31 (AF3628; R&D Systems), and CD206 (GTX42265; GeneTex).

Techniques: Flow Cytometry, Control