pe labeled anti cx3cr1  (Revvity Signals)

Journal: The Journal of Experimental Medicine

Article Title: KLF family members control expression of genes required for tissue macrophage identities

doi: 10.1084/jem.20240379

Figure Lengend Snippet: Impaired development of LCMs in myeloid-specific KLF2-deficient mice. (A) Flow cytometry of cells in peritoneal cavity lavage from the indicated mice, gated on live, single, CD3 − B220 − CD19 − cells. Numbers adjacent to gates indicate the percentage of total live events for each gate. (B) Bar graphs depicting the percentage of total live cells and total number of cells for the following populations (representative gates shown in A) as measured by flow cytometry: CD11b + F4/80 + , SCM (CD11b + F4/80 + MHCII hi DNAM-1 hi ), LCMs (CD11b + F4/80 + MHCII − DNAM-1 − ), ICAM2 + GATA6 + LCM (CD11b + F4/80 + MHCII − DNAM-1 − ICAM2 + GATA6 + ), and transitional (CD11b + F4/80 + MHCII mid DNAM-1 mid ). (C) Dimensionality reduction via UMAP of combined CD11b + F4/80 + cells from LysM +/+ Gata6 fl/fl and LysM +/+ Klf2 fl/fl (gated together as LysM +/+ controls), LysM Cre/+ Gata6 fl/fl , or LysM Cre/+ Klf2 fl/fl mice, downsampled to normalize cells per genotype. The resulting projection was overlayed with supervised gates for LCMs, transitional, and SCMs as shown in A. The upper panel shows all cells, while the lower panels show only cells from the indicated genotypes. (D) Heatmaps of defining marker expression for SCM, LCM, and transitional cell clusters overlaid onto the UMAP projections from C. Data in A are from one experiment representative of six independent experiments; data in B are combined from six independent experiments, LysM +/+ Gata6 fl/fl ( n = 9), LysM Cre/+ Gata6 fl/fl ( n = 6), LysM +/+ Klf2 fl/fl ( n = 5), LysM Cre/+ Klf2 fl/fl ( n = 7), CX3CR1-Cre − Klf2 fl/fl ( n = 4), and CX3CR1-Cre + Klf2 fl/fl ( n = 5). Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction. Asterisks denote: ****P < 0.0001, ***P = 0.0006, **P = 0.0021, and *P = 0.033.

Article Snippet: The following mice were used in this study: C57BL/6J (000664), B6.SJL (B6.Ptprc a Pep c /BoyJ, 002014), JaxBoy (C57BL/6J-Ptprcem6Lutzy/J, 033076), LysM Cre (Lyz2Cretm1[Cre]Ifo, 004781), Klf2 fl/fl (gift from Jerry B. Lingrel, University of Cincinnati, Cincinnati, OH, USA; deceased), LysM Cre Gata6 fl/fl (gift from Paul Kubes, University of Calgary, Calgary, Canada), LysM Cre Klf4 fl/fl (floxed Klf4 allele: MMRRC 29877), CX3CR1-Cre (Tg[Cx3cr1-cre]MW126Gsat/Mmucd, MMRRC: 036395-UCD), CD11c-Cre Klf4 flfl (Tg[Itgax-cre]1-1Reiz/J, 008068, cross generated in-house), GFP-KLF2 (B6[C]-Klf2tm1.1Khog/JmsnJ, gift from Stephen Jameson and Kristin Hogquist labs, University of Minnesota, Minneapolis, MN, USA), and CD45.1 × CD45.2 F1 (generated in our colony by crossing C57BL/6J to B6.SJL [B6.Ptprc]).

Techniques: Flow Cytometry, Marker, Expressing

Journal: Journal of Neuroinflammation

Article Title: Centripetal migration and prolonged retention of microglia promotes spinal cord injury repair

doi: 10.1186/s12974-025-03411-9

Figure Lengend Snippet: Co-administration of CX3CL1 and M-CSF enhances centripetal migration and prolonged retention of microglia after SCI. ( A ) Schematic diagram illustrating in situ injection of 2 µl CX3CL1 and M-CSF (CX3CL1 + M-CSF group), 1 µl CX3CL1 (CX3CL1 group), 1 µl M-CSF (M-CSF group) or 1 µl PBS (PBS group) into the epicenter immediately after SCI in WT and CX3CR1 GFP CCR2 RFP mice. Histological analysis was performed at 28 dpi. ( B ) Immunofluorescence staining of GFAP (green) and Fascin-1 (red) showing an augmented presence of Fascin-1 + microglia at the injury sites in the CX3CL1 + M-CSF group compared to the CX3CL1 and M-CSF groups at 28 dpi of WT mice. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. ( C ) Quantification of the number of Fascin-1 + cells in the epicenter at 28 dpi ( n = 4 mice per group). ( D ) Immunofluorescence staining of Mac-2 (red) and DAPI (blue) in sagittal sections from the CX3CL1 + M-CSF, CX3CL1 and M-CSF groups at 28 dpi of WT mice. ( E ) Quantification of the percentage of Mac-2 + area within the spinal cord segment spanning the injured core at 28 dpi ( n = 4 mice per group). ( F ) Representative immunofluorescence images of RFP (red) and GFP (green) in sagittal sections of the injured spinal cord from the CX3CL1 + M-CSF and PBS groups at 28 dpi of CX3CR1 GFP CCR2 RFP mice. ( G ) Quantification of the percentage of RFP + area within the spinal cord segment spanning the injured core at 28 dpi ( n = 3 mice per group). The data are presented as means ± SEM (error bars). **** P < 0.0001, the CX3CL1 + M-CSF group versus the CX3CL1, M-CSF and PBS groups. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests ( C and E ), or Student’s two-tailed unpaired t-test ( G ). Scale bars = 100 μm ( B and F ), 200 μm ( D ), 20 μm ( ROI )

Article Snippet: The CX3CRl GFP CCR2 RFP mice [ ] and CX3CR1 −/− (CX3CR1 GFP/GFP ) mice [ ] at age of 8 weeks were obtained from Jackson Laboratory (catalog no. 032127 and 005582).

Techniques: Migration, In Situ, Injection, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Centripetal migration and prolonged retention of microglia promotes spinal cord injury repair

doi: 10.1186/s12974-025-03411-9

Figure Lengend Snippet: The beneficial effects of combined administration of CX3CL1 and M-CSF are specifically blocked in CX3CR1 −/− mice. (A) Schematic diagram illustrating in situ injection of 2 µl of CX3CL1 and M-CSF into the epicenter immediately after SCI in CX3CR1 −/− (CX3CR1 GFP/GFP ) and CX3CR1 +/− (CX3CR1 +/GFP ) mice. Histological analysis was performed at 28 dpi. (B) Representative immunofluorescence images of GFAP (red) and GFP (green) from sagittal sections of the injured spinal cord of CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. (C) Quantification of the number of GFP + microglia in the epicenter of CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi ( n = 3 mice per group). (D) Representative images of sections of the injured spinal cord of CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi, stained with antibodies against GFAP (green), PDGFRβ (red) and NeuN (purple). (E and F) Quantification of the percentage of GFAP − area (E) and PDGFRβ + area (F) within the spinal cord segment spanning the injured core in CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi ( n = 3 mice per group). (G) Quantification of NeuN + neurons in Z1–Z3 zones adjacent to central lesion core in CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi ( n = 3 mice per group). (H) Immunofluorescence staining of 5-HT (red) and DAPI (blue) in sagittal sections of the injured spinal cord from CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi. White arrowheads indicate the lesion site. (I) Quantification of 5-HT density within the spinal cord segment spanning the injured core in CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi. ( n = 3 mice per group). (J) Time course of functional recovery of CXCR1 −/− and CX3CR1 +/− mice assessed by the BMS score after the combined treatment of CX3CL1 and M-CSF post-SCI ( n = 6 mice per group). (K) Footprint analysis performed at 28 dpi reveals improved functional recovery in the CX3CR1 +/− mice after the combined treatment of CX3CL1 and M-CSF. (L) Quantification of the footprint analysis parameters (stride length, stride width, and paw rotation) in CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi ( n = 6 mice per group). The data are presented as means ± SEM (error bars). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001, the CX3CR1 +/− mice versus the CX3CR1 −/− mice. Statistical analysis was performed using two-way ANOVA (G and J) followed by Bonferroni post hoc tests’, or Student’s two-tailed unpaired t-test ( C , E , F , I and L ). Scale bars = 200 μm ( D and H ), 100 μm ( B ), 20 μm ( ROI )

Article Snippet: The CX3CRl GFP CCR2 RFP mice [ ] and CX3CR1 −/− (CX3CR1 GFP/GFP ) mice [ ] at age of 8 weeks were obtained from Jackson Laboratory (catalog no. 032127 and 005582).

Techniques: In Situ, Injection, Immunofluorescence, Staining, Functional Assay, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Centripetal migration and prolonged retention of microglia promotes spinal cord injury repair

doi: 10.1186/s12974-025-03411-9

Figure Lengend Snippet: Co-administration of CX3CL1 and M-CSF may exert beneficial effects by enhancing SYK levels in CX3CR1 + microglia and reducing foamy cell recruitment after SCI. ( A ) Immunofluorescence staining of SYK (green) and DAPI (blue) in sagittal sections from the CX3CL1 + M-CSF, CX3CL1, M-CSF, Control and PBS groups at 28 dpi. ROI represents the high-magnification image within the dotted box on the left. Asterisks show the injured core. ( B ) Quantification of the percentage of SYK + area within the spinal cord segment spanning the injured core at 28 dpi ( n = 4 mice per group). ( C ) Lipid accumulation was determined by ORO staining at 28 dpi. ( D ) The quantification of the ratio of the mean density of ORO signal to the lesion area within the spinal cord segment spanning the injured core at 28 dpi ( n = 3 mice per group). ( E ) Representative immunofluorescence images of SYK (red) and GFP (green) in sagittal sections of the injured spinal cord from CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi. These mice were treated with an in situ injection of 2 µl of CX3CL1 and M-CSF into the epicenter immediately after SCI. ( F ) Quantification of the percentage of SYK + area within the spinal cord segment spanning the injured core in CX3CR1 −/− and CX3CR1 +/− mice at 28 dpi ( n = 3). The data are presented as means ± SEM (error bars). * P < 0.05 and ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests ( B and D ), or Student’s two-tailed unpaired t-test ( F ). Scale bars = 200 μm ( A , C and E ), 20 μm ( ROI )

Article Snippet: The CX3CRl GFP CCR2 RFP mice [ ] and CX3CR1 −/− (CX3CR1 GFP/GFP ) mice [ ] at age of 8 weeks were obtained from Jackson Laboratory (catalog no. 032127 and 005582).

Techniques: Immunofluorescence, Staining, Control, In Situ, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Two-Stage CD8 + CAR T-Cell Differentiation in Patients with Large B-Cell Lymphoma

doi: 10.1101/2025.03.05.641715

Figure Lengend Snippet: ( a , b ) UMAP depicting single-cell transcriptomes of peripheral blood CD8 + T cells colored by cell cluster ( a ) or density contours of each cluster ( b ). The exhausted-like EM cluster is located on the lower half of the UMAP. ( c ) Density maps depicting expression levels of major T-cell genes and proteins, divided into categories. In the “receptor” category, proteins are placed directly beneath the corresponding gene. ( d ) Flow plots for validating transcriptomic data. Plots depict expression of CD39 and CD57 (top row) or CX3CR1 (bottom row) in CD8 + CAR T cells at each timepoint from P2. Exhausted-like EM CAR T cells were expected to be T exp -specific with high CD39 and low CD57/CX3CR1 expression. ( e , f ) Heat map depicting normalized expression of major gene sets from Wherry et al. ( e ). Expression of two of the gene sets were depicted as violin plots ( f ), ordered by decreasing expression level per cluster. Expression levels were compared to that of the cluster with highest expression via Wilcoxon Rank-Sum test, with p-values adjusted for multiple hypotheses testing using the Benjamini-Hochberg method, whereby **** indicates p<0.0001, *** indicates p<0.001. ( g ) Volcano plot depicting differentially expressed genes between EM and exhausted-like EM CD8 + CAR T cells. Genes were colored according to direction of upregulation. ( h ) Enrichment plots for select gene sets differentially expressed between EM and exhausted-like EM CD8 + CAR T cells.

Article Snippet: Then, cells were incubated for 30 minutes at 4°C in the dark with a staining solution containing AF647-labeled CD19-tetramers (3 nM final concentration), BV421-labeled anti-CD3ε (clone SK7, BioLegend, 344833), AF488-labeled anti-CD8α (clone SK1, BioLegend, 344716), PE-labeled anti-CX3CR1 (clone 2A9-1, BioLegend, 341603), PE/Cy7-labeled anti-CD39 (clone A1, BioLegend, 328211), and BV605-labeled anti-CD57 (clone QA17A04, BioLegend, 393303).

Techniques: Expressing