cx 3 cr1 gfp  (Jackson Laboratory)

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: CAM, PEM and pericytes are present at all branch orders of capillaries. (a) Representative image stack (20–75 μm depth, average intensity projection) of FITC dextran filled vessels in the somatosensory cortex of an NG2DsRed × CX 3 CR1 +/GFP mouse imaged in vivo using 2PLSM. Pericytes are labeled magenta, and microglia and vessels are green. Insets correspond to boxes labeled 1–8 in main panel. (1) Penetrating arteriole (0th order) branching off to form a capillary (first order). (2–7) Higher order capillaries branching. (8) Seventh order capillary converging on the ascending venule. (b, c) Percentage of total CAM, PEM and pericytes that are located (b) at different branch orders of the vascular tree, and (c) at vessel junctions at different branch orders of the vascular tree ( n = 5, four male and one female). For all graphs, gray circles represent males and white circles represent females. Data presented as mean ± SD.

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: In Vivo, Labeling

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: Pericytes can gain or lose PEM over 28 days and vessel width is increased beneath pericyte and microglia soma. (a) Representative in vivo 2PLSM images of a PEM that maintains position for 28 days in the somatosensory cortex of an NG2DsRed × CX 3 CR1 +/GFP mouse. (b) Representative images of a PEM in position for 4 days that is no longer in place on day 7 or 28 of imaging. (c) Representative images of a pericyte with no adjacent PEM on day 0 or 4 but gains a PEM on day 7. (a–c) For all images pericytes are magenta, microglia are green, and images are average intensity projections. White arrowheads highlight a PEM and white asterisks indicate the absence of a PEM. (d, e) Percentage of (d) 32 PEM, identified on day 0 of imaging, that maintain position adjacent to a pericyte on days 4, 7 and 28 of imaging and (e) total pericytes with a PEM per mouse on days 0, 4, 7 and 28 ( n = 6, three male and three female). Data in (e) analyzed using a repeated measures one‐way ANOVA (day of imaging F (2.1, 10.4) = 1.8, p = .2137), with Tukey's post hoc test. (f) Representative images indicating locations of vessel width measurement in in vivo 2PLSM images derived from NG2DsRed × CX 3 CR1 +/GFP mice, following administration of the vessel lumen marker FITC‐dextran. White arrowheads indicate the midpoint below the cell soma. (g) Quantification of vessel width (μm) of vessel only (VO), and vessels with a pericyte (P), CAM or a pericyte with a PEM (150, 124, 90 and 124 vessels were measured, respectively, across n = 8 mice, five male and three female). Data presented as average per animal with a minimum of four vessel measurements at VO, P, CAM and PEM per animal. Comparisons were made with a Friedman's test (Friedman statistic = 14.85, p = .0019), with Dunn's post hoc test. (h–i) Quantification of vessel width (μm) of vessels with a pericyte where a PEM was (h) gained or (i) lost ( n = 3 mice, 1 male and 2 female). Data presented as average per animal with a minimum of two vessel measurements. Comparisons made with a paired parametric t ‐test. For all graphs, gray circles represent males and white circles represent females. Data presented as mean ± SD. * p < .05; ** p < .01.

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: In Vivo, Imaging, Derivative Assay, Marker

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: A subset of microglia are directly adjacent to pericytes. (a) Representative example of a capillary‐associated microglia (CAM), in the somatosensory cortex, extending fine processes to contact other DAPI‐positive nuclei. (b) Representative example of two pericyte‐associated microglia (PEM), in the hippocampus, with the bottom PEM morphologically curved around a pericyte. (a, b) For all images: NG2DsRed‐positive pericytes (magenta), CX 3 CR1 +/GFP ‐positive microglia (green), IB4‐labeled vessels (white) and DAPI‐labeled nuclei (blue). Each fluorescent channel alone is located below the main image. All images were derived from 12‐week‐old NG2DsRed × CX 3 CR1 +/GFP mice using confocal microscopy. (c) Schematic of region analyzed within the somatosensory cortex of NG2DsRed × CX 3 CR1 +/GFP mice (Bregma −1.5 mm [Allen Reference Atlas, ]). (d, e) Quantification of (d) CAM and (e) PEM in the somatosensory cortex of male ( n = 5) and female ( n = 5) mice. Data compared with an unpaired parametric t ‐test. (f) The predicted percentage of PEM when running simulations with microglia and pericyte densities derived from 10 different biological replicates, compared to the actual PEM percentage from those same 10 somatosensory biological replicates (biological data derived from [e]). Data compared with a Wilcoxon test. Lines represent paired data. (g) Schematic of cranial window location in NG2DsRed × CX 3 CR1 +/GFP mice with blood vessels used as landmarks. (h) Representative 30 μm thick projection image of NG2DsRed‐positive pericytes (magenta), CX 3 CR1 +/GFP ‐positive microglia (green) and FITC‐dextran‐positive vessel lumen (green) in layers II/III of the somatosensory cortex of adult NG2DsRed × CX 3 CR1 +/GFP mice imaged using 2PLSM. Dashed boxes highlighting a CAM and PEM are magnified in panels to the right. (i–k) Quantification of the percentage of microglia that are (i) CAM, (j) PEM and (k) the percentage of CAM that are PEM, in layers II/III of the somatosensory cortex of male ( n = 3) and female ( n = 3) mice. (l) Representative image of PDGFRβ‐positive pericyte (magenta), IBA1‐positive microglia (green), UEA‐1‐labeled vessels (white) and DAPI‐labeled nuclei (blue) from the SFG of a human control brain (93 y.o. female). A CAM and PEM are highlighted by white arrows. For all graphs, gray circles represent males and white circles represent females. Data presented as mean ± SD. ** p < .01.

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: Labeling, Derivative Assay, Confocal Microscopy, Control

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: Microglia can interact with pericytes with or without AQP4‐positive astrocyte endfeet. (a) Representative example of a pericyte (magenta) with no associated PEM exhibiting strong labeling by AQP4 (white). (b) Representative example of a pericyte (magenta) that is not associated with a PEM, but lacking AQP4 (white) labeling. (c) Representative example of a pericyte (magenta) associated with a PEM (green). The pericyte is exhibiting strong labeling by AQP4 (white). (d) Representative example of a pericyte (magenta) associated with a PEM (green). The pericyte is lacking AQP4 (white) labeling. (e) Schematic of region analyzed in 12‐week‐old NG2DsRed × CX 3 CR1 +/GFP mice (~Bregma −1.10 mm [Allen Reference Atlas, ]). (f) Quantification of AQP4 fluorescence intensity in regions overlaying pericytes with, or without, an associated PEM ( n = 5, two males, three females). Data compared with a paired parametric t ‐test. (g) AQP4 fluorescence intensity around individual pericytes with, or without, an associated PEM ( n = 5, two males, three females). This data was used to derive the averages in (f). Data are presented as mean ± SD. For all images: NG2DsRed‐positive pericytes (magenta), CX 3 CR1 +/GFP ‐positive microglia (green), AQP4‐labeled astrocyte endfeet (white) and DAPI‐labeled nuclei (blue) are shown. Images showing each fluorescent channel alone are to the right of the main image.

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: Labeling, Fluorescence

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: Microglia can directly associate with the basement membrane covering pericytes. (a) Representative example of a pericyte‐associated microglia extending a process around a pericyte at the border of the somatosensory and motor cortex (~Bregma −1.5 mm). This representative image shows the basement membrane between a microglial cell body and process and a pericyte soma. NG2DsRed‐positive pericyte (magenta), CX 3 CR1 +/GFP ‐positive microglia (green), IB4‐labeled vessels (white) and DAPI‐labeled nuclei (blue). (b) Magnification of dashed box in (a), rotated to illustrate the presence of the basement membrane (IB4, white) between the microglia (green) and pericyte (magenta). All images were derived from 12‐week‐old NG2DsRed × CX 3 CR1 +/GFP mice using confocal microscopy. Individual channels are shown in Supplementary Figure . (c–e) Representative serial section transmission EM images of a microglia (green) associating with a pericyte (orange, magenta and cyan) on a capillary (light‐purple), through a stack starting at a z‐depth of (c) 0 nm, (d) 800 nm and (e) 1200 nm. Left column shows raw images, center column shows cellular components pseudo colored. Dashed boxes in left and center column images are magnified in the bottom right corner of each image. The EM images in the left column show an endothelial cell (bottom left of all images) segregated from the pericyte by a basement membrane. In the center column these cells have not been segmented separately, so have been pseudo colored the same (cyan). Although we found examples of pericytes and endothelial cells correctly segmented in this dataset, it is common to find mistakes in the segmentation, as observed by others (Bonney et al., ). Black arrowheads indicate where the microglia cell body is in direct contact with the basement membrane covering the pericyte cell body. White arrowheads indicate where astrocyte endfeet are observable between the two cells. The right column shows 3D reconstructions of the pericyte and microglia; top right is zoomed out with all colors highlighted, second top right is a magnified view of the top right image and bottom right is with the vessels removed. Coordinates are: X : 365279, Y : 175038, Z : 23443. Images generated from https://www.microns‐explorer.org/cortical‐mm3 .

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: Membrane, Labeling, Derivative Assay, Confocal Microscopy, Transmission Assay, Generated

Journal: Glia

Article Title: Microglia directly associate with pericytes in the central nervous system

doi: 10.1002/glia.24371

Figure Lengend Snippet: CX 3 CR1 knock‐out does not alter the proportion of microglia that are PEM or CAM and does not alter vessel width at PEM and CAM in 12‐week‐old mice. (a, b) Representative images of (a) NG2DsRed × CX 3 CR1 +/GFP and (b) NG2DsRed × CX 3 CR1 GFP/GFP mice showing that PEM are present in both mouse lines (white arrows). For each image: NG2DsRed‐positive pericytes (magenta), CX 3 CR1 +/GFP ‐positive microglia (green), IB4 (white) and DAPI‐labeled nuclei (blue) are shown. Images showing each fluorescent channel alone are to the right of the main image. (c–f) Quantification of the percentage of microglia that are (c) CAM and (d) PEM, and width (μm) of IB4 labeled capillaries at sites of (e) CAM and (f) PEM in the somatosensory cortex (c–f, Bregma −1.5; n = 5 per group, two males and three females for NG2DsRed × CX 3 CR1 +/GFP , three males and two females for NG2DsRed × CX 3 CR1 GFP/GFP ). Data compared with an unpaired parametric t ‐test. For all graphs, gray circles represent males and white circles represent females. Data presented as mean ± SD.

Article Snippet: Hemizygote NG2DsRed transgenic mice (The Jackson Laboratory stock #008241) were backcrossed onto a C57BL/6J background and crossbred with CX 3 CR1 GFP/GFP transgenic mice (The Jackson Laboratory stock #005582, C57BL/6J background) to produce NG2DsRed × CX 3 CR1 +/GFP or NG2DsRed × CX 3 CR1 GFP/GFP mice.

Techniques: Knock-Out, Labeling

Journal: JCI Insight

Article Title: Acute high-fat diet impairs macrophage-supported intestinal damage resolution

doi: 10.1172/jci.insight.164489

Figure Lengend Snippet: ( A and B ) Il10 gene expression from cecum ( n = 4 LFD and HFD, n = 5 LFD DSS and HFD DSS) ( A ) and intestinal ( B ) CX 3 CR1 + macrophages from LFD- and HFD-fed DSS-treated mice ( n = 4 samples/group). ( C ) Il10 gene expression in control (Ctrl), oleic acid (OA), or palmitic acid (PA) pretreated macrophages after exposure to dead neutrophils (2 pooled experiment with n = 4 technical replicates/group). ( D ) Body weight change in HFD-fed DSS-treated mice after hydrodynamic delivery of control or IL-10–producing plasmid ( n = 6 control and n = 9 IL-10 mice/group). ( E – L ) measurements in cecum of mice in D ( n = 6 mice per group). ( E and F ) Representative H&E staining and blinded colitis score. ( G and H ) Representative staining and quantification of Ki67 + proliferating cells. ( I and J ) Occludin (Ocln) and Zo1 (Tjp1) expression. ( K and L ) Representative Alcian blue/PAS staining and quantification of goblet cells. For all imaging, the average of 3 HPF images was taken per mouse. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001. Statistical comparisons were performed using Student’s t test ( B , D , F , H–J , and L ) and 1-way ANOVA with Tukey’s post hoc test ( A and C ), and if not indicated, a comparison is not significant. Scale bar: 100 μm.

Article Snippet: Male C57BL/6J (stock no. 000664), CX 3 CR1-GFP/+ (stock no. 005582), CX 3 CR1-CreERT2 (stock no. 021160), and Lgr5-EGFP-IRES-creERT2 (stock no. 008875) mice were purchased from The Jackson Laboratory.

Techniques: Gene Expression, Control, Plasmid Preparation, Staining, Expressing, Imaging, Comparison

Journal: Journal of Ovarian Research

Article Title: Fractalkine receptor is expressed in mature ovarian teratomas and required for epidermal lineage differentiation

doi: 10.1186/1757-2215-6-57

Figure Lengend Snippet: CX 3 CR1 is required for keratinocytes differentiation. (A) Expression of CX 3 CR1 in parental PA-1 cell line and that stably transfected with either CX 3 CR1 or scrambled shRNA constructs was examined with Western blot. Actin served as a loading control. CX 3 CR1 expression levels were quantified using digital densitometry and normalized to the levels of actin expression. (B) Expression of cytokeratin 14 in PA-1 cells stably transfected with either CX 3 CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with immunocytochemistry. CK14 in PA-1 was probed with anti-CK14 and Alexa555-conjugated anti-rabbit antibodies, and DNA was stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI); CK14 – red, DNA – blue. Images were taken using 5× magnification on the objective. The histogram demonstrates the average intensity of CK14 signal across the field as determined by the Zeiss AxioVision software. Data is an average of three independent experiments. * p < 0.05. (C) Expression of cytokeratins 14 and 18 in PA-1 cells stably transfected with either CX 3 CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with Western blot. β-Tubulin served as a loading control. The histogram shows CK14 and CK18 expression levels. Expression of CK18 in CX 3 CR1shRNA-transfected PA-1 cells was arbitrarily set as 1 and expression of both CK14 and CK18 in other conditions was calculated accordingly. The data represent a typical Western blot image, and quantitative analysis was performed using three independently performed experiments. * p < 0.05. (D) Expression of CX 3 CL1 in SKOV-3 and PA-1 cell lines was examined with Western blot. Actin served as a loading control. SKOV-3 cell line was used as a positive control.

Article Snippet: GFP-tagged CX 3 CR1 shRNA and scrambled shRNA constructs were obtained from Origene Technologies (Rockville, MD).

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Construct, Western Blot, Recombinant, Immunocytochemistry, Staining, Software, Positive Control

Journal: Journal of Ovarian Research

Article Title: Fractalkine receptor is expressed in mature ovarian teratomas and required for epidermal lineage differentiation

doi: 10.1186/1757-2215-6-57

Figure Lengend Snippet: CX 3 CR1 is required for keratinocytes differentiation. (A) Expression of CX 3 CR1 in parental PA-1 cell line and that stably transfected with either CX 3 CR1 or scrambled shRNA constructs was examined with Western blot. Actin served as a loading control. CX 3 CR1 expression levels were quantified using digital densitometry and normalized to the levels of actin expression. (B) Expression of cytokeratin 14 in PA-1 cells stably transfected with either CX 3 CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with immunocytochemistry. CK14 in PA-1 was probed with anti-CK14 and Alexa555-conjugated anti-rabbit antibodies, and DNA was stained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI); CK14 – red, DNA – blue. Images were taken using 5× magnification on the objective. The histogram demonstrates the average intensity of CK14 signal across the field as determined by the Zeiss AxioVision software. Data is an average of three independent experiments. * p < 0.05. (C) Expression of cytokeratins 14 and 18 in PA-1 cells stably transfected with either CX 3 CR1 or scrambled shRNA constructs treated with recombinant BMP-4 or vehicle, as indicated, was examined with Western blot. β-Tubulin served as a loading control. The histogram shows CK14 and CK18 expression levels. Expression of CK18 in CX 3 CR1shRNA-transfected PA-1 cells was arbitrarily set as 1 and expression of both CK14 and CK18 in other conditions was calculated accordingly. The data represent a typical Western blot image, and quantitative analysis was performed using three independently performed experiments. * p < 0.05. (D) Expression of CX 3 CL1 in SKOV-3 and PA-1 cell lines was examined with Western blot. Actin served as a loading control. SKOV-3 cell line was used as a positive control.

Article Snippet: PA-1 cells were cultured to 80% confluence and transfected with either CX 3 CR1-specific GFP-tagged shRNA construct or scrambled GFP-tagged shRNA construct (Origene Technologies) using DharmaFECT according to the manufacturer’s instructions.

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Construct, Western Blot, Recombinant, Immunocytochemistry, Staining, Software, Positive Control