Journal: Nature Communications

Article Title: Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction

doi: 10.1038/s41467-022-35331-0

Figure Lengend Snippet: a Principal components analysis (PCA) of transcriptomes of sham-operated (Sham, n = 3), GI254023X (A10i, n = 4) and DMSO ( n = 4) treated mice 3 days after myocardial infarction. Principal components were computed for each sample using gene expression. The first two principal components (PC1 and PC2) are shown. The percent of variance explained by each component is reported in parentheses. b Volcano plot representing the expression changes of all genes. Significantly down- and upregulated genes (FDR < 0.01) are colored navy and magenta, respectively. Genes that do not show significant expression changes are colored gray. Examples of neutrophil chemotaxis-associated genes are labeled with gene names. P -values were calculated using the R packages DESeq2 R (v1.30.1) and IHW (1.18.0) . Functional annotation clustering for the gene ontology (GO) terms c biological process and d molecular function of genes with significantly decreased expression upon A10i treatment (Fisher exact test with Benjamini-Hochberg posttest). e , f Gene set enrichment analysis of genes with significantly decreased expression upon A10i treatment performed on GO terms. Gene set enrichment analysis was performed using the GSEA tool (v4.1.0) of the Broad Institute and the DESeq2 R package (v1.30.1) . g In-depth analysis of the significantly downregulated genes of the indicated functional annotation clusters. h Quantification of CXCL1 ( n = 4 vs. 5, P = 0.380952), CXCL5 ( n = 8 vs. 8, P = 0.633877), CXCL16 ( n = 10 vs. 8, P = 0.572604), CX3CL1 ( n = 9 vs. 9, P = 0.00399), IL-1β ( n = 8 vs. 8, P = 0.000155) and IL-6 ( n = 6 vs. 5, P = 0.452381), serum levels in A10i and DMSO treated mice 3 days after MI (mean ± SEM, ns not significant, ** P < 0.01, *** P < 0.001, two-tailed Mann–Whitney test). i Western blot analysis and j , k quantification of j CX3CL1 and k IL-1β in heart tissue lysates of sham-operated (Sham) and LAD-ligated (MI) mice 3 days after infarction ( n = 5, mean ± SEM, ns not significant, * P < 0.05, ** P < 0.01, Kruskal–Wallis test with Dunn’s posttest, exact P -values are provided in the Source Data file). l Western blot analysis and m quantification of CX3CL1 in heart tissue lysates from patients with ischemic cardiomyopathy (ICM) and non-failing controls (NF) ( n = 6, mean ± SEM, ** P = 0.0031, two-tailed t test). Source data are provided as a Source Data file.

Article Snippet: Specific siRNAs against murine CX3CL1 (SR426672A, SR426672C) and unspecific control siRNA (SR300004) were obtained from Origene.

Techniques: Gene Expression, Expressing, Chemotaxis Assay, Labeling, Functional Assay, Two Tailed Test, MANN-WHITNEY, Western Blot

Journal: Nature Communications

Article Title: Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction

doi: 10.1038/s41467-022-35331-0

Figure Lengend Snippet: a Immunofluorescence images and b quantification of CX3CL1, cardiac Troponin T (cTnT, cardiomyocytes, CM) and CD31 (endothelial cells, EC) stained heart tissue sections of sham-operated (Sham) and LAD-ligated (MI) mice 14 days after surgery. Nuclei are stained with DAPI. Representative images are shown. Scale bar, 20 µm. n = 4 per group, mean ± SEM, ns not significant, two-tailed Mann–Whitney test with Dunn’s posttest. c Co-immunoprecipitation of endogenous ADAM10 (A10) and CX3CL1 from whole-cell lysates of HL-1 cells. Representative western blots are shown ( n = 4). IgG, immunoglobulin G as a control. WCL, whole-cell lysate. d Western blot analysis of CX3CL1 and e quantification of CX3CL1 expression ( n = 6 per group, mean ± SEM, ns not significant, ** P < 0.01, *** P < 0.001, one-way ANOVA with Tukey’s posttest) as well as f ectodomain shedding (CX3CL1 levels in supernatant) in normoxic as well as GI254023X (A10i) and DMSO treated hypoxic (1% O 2 ) mouse cardiomyocytes (HL-1) ( n = 3 per group, mean ± SEM, ns not significant, ** P < 0.01, Kruskal–Wallis test with Dunn’s posttest, exact P -values are provided in the Source Data file). g Treatment scheme. HL-1 cells were cultured for 3 h under normoxic or hypoxic conditions in parallel to A10i or DMSO treatment in combination with unspecific control siRNA (siC) or CX3CL1 depletion (siCX3CL1). Supernatants were used in the lower chamber and 3 × 10 5 freshly isolated mouse bone marrow derived neutrophils or macrophages were seeded in the upper chamber of transwell plates (8 µm pore size) to determine migration capacity. The scheme was created using Servier Medical Art (available online: http://smart.servier.com ). h Western blot analysis and i quantification of CX3CL1 expression in HL-1 cells treated for 48 h with unspecific control siRNA (siC) or CX3CL1-specific siRNA (siCX3#1 and siCX3#2). Representative western blots ( n = 3 per group, mean ± SEM, ** P < 0.01, Kruskal–Wallis test with Dunn’s posttest) are shown. Exact P -values: siC vs. siCX3#1, 0.0043. siC vs. siCX3#2, 0.005. Transwell migration assays of bone marrow derived j neutrophils and k macrophages using the supernatants of HL-1 cells that were cultured under hypoxic conditions in parallel to A10i or DMSO treatment in combination with unspecific control siRNA (siC) or CX3CL1 depletion (siCX3#1 and CX3#2) as chemoattractant ( n = 3 per group, mean ± SEM, ns not significant, ** P < 0.01, Kruskal–Wallis test with Dunn’s posttest). Source data and exact P -values are provided in the Source Data file.

Article Snippet: Specific siRNAs against murine CX3CL1 (SR426672A, SR426672C) and unspecific control siRNA (SR300004) were obtained from Origene.

Techniques: Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Immunoprecipitation, Western Blot, Control, Expressing, Cell Culture, Isolation, Derivative Assay, Pore Size, Migration

Journal: Nature Communications

Article Title: Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction

doi: 10.1038/s41467-022-35331-0

Figure Lengend Snippet: a – c Echocardiographic assessment of fractional area shortening, ejection fraction and left ventricular end-systolic interior diameter in 8-week-old ADAM10 fl/fl (WT) and (αMHC-Cre) ADAM10 KO mice under a basal conditions (WT, n = 7, KO, n = 8, mean ± SEM, ns not significant) as well as b 3 days (WT, n = 8, KO, n = 9, mean ± SEM, ns not significant, * P < 0.05, two-tailed t test) and c 14 days after myocardial infarction (WT, n = 6, KO, n = 9, mean ± SEM, ns not significant, * P < 0.05, two-tailed t test, exact P -values are provided in the Source Data file). d Representative end-systolic B-mode and M-mode echocardiograms of ADAM10 WT and ADAM10 KO mice 14 days after myocardial infarction. e Western blot analysis and f quantification of ADAM10, CX3CL1 and IL-1β expression in heart tissue lysates of ADAM10 WT and ADAM10 KO mice 3 days after myocardial infarction ( n = 6, mean ± SEM, ns not significant, * P < 0.05, ** P < 0.01, two-tailed t test). Exact P -values: ADAM10, 0.0159. CX3CL1, 0.0084. IL-1β, 0.0317. g Analysis of leukocyte counts in blood samples of ADAM10 WT ( n = 8) and ADAM10 KO ( n = 7) mice (mean ± SEM, ns not significant, * P < 0.05, two-tailed t test). Exact P -values: Neutrophils, 0.0404. Eosinophils, 0.7886. Monocytes, 0.3373. Lymphocytes, 0.751. h Analysis of leukocyte counts in the infarcted area/ infarct border zone of ADAM10 WT ( n = 8) and ADAM10 KO ( n = 7) mice (mean ± SEM, ns not significant, * P < 0.05, two-tailed two-tailed t test). Exact P -values: Neutrophils, 0.0214. Eosinophils, 0.176. Monocytes, 0.639. Macrophages, 0.6064. Source data are provided as a Source Data file.

Article Snippet: Specific siRNAs against murine CX3CL1 (SR426672A, SR426672C) and unspecific control siRNA (SR300004) were obtained from Origene.

Techniques: Two Tailed Test, Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption.

doi: 10.4049/jimmunol.0803627

Figure Lengend Snippet: FIGURE 4. Close contacts with CX3CL1-positive osteoblasts and CX3CR1-positive osteoclast precursors in vivo. A, FACS analysis. Primary osteoblasts isolated from neonatal mouse calvariae were stained for surface CX3CL1. FACS profiles by control hamster IgG (shaded area) and ham- ster anti-CX3CL1 mAb (clone 5H8–4; blue line) are shown. B, Immuno- histochemical analysis of human bone tissue. Osteoblasts colocalized with the bone are clearly positive for CX3CL1. Arrowheads indicate osteoblasts and asterisks indicate bone. Original magnification, 200. Representative results from experiments repeated at least three times are shown. C, Close contact of CX3CL1-positive osteoblasts (white arrowheads) and CX3CR1- positive osteoclast precursors (black arrows) by double staining for CX3CL1 (brown) and CX3CR1 (blue). Asterisks indicate bone. Original magnification, 1000.

Article Snippet: Effects of CX3CL1 on differentiation and CX3CR1 expression of osteoclast precursors Osteoclast precursors (nonadherent mouse bone marrow cells) were cultured in -MEM supplemented with 10% FBS with or without 10 ng/ml murine recombinant chemokine domain CX3CL1 (10 nM; R&D Systems).

Techniques: In Vivo, Isolation, Staining, Control, Double Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption.

doi: 10.4049/jimmunol.0803627

Figure Lengend Snippet: FIGURE 3. No direct effect of CX3CL1 stimulation on differentiation and CX3CR1 expression of osteoclast precursors in vitro. A, Osteoclast precursors derived from mouse bone marrow cells were cultured in the presence of recombinant RANKL (100 ng/ml; a) or recombinant CX3CL1 (10 nM; b) for 2 days and stained for TRAP. Asterisks indicate TRAP- positive osteoclasts. B, RT-PCR of osteoclast precursors cultured in the presence of recombinant CX3CL1 (10 nM) for 2 days was performed for CX3CR1genes. GAPDH served as the loading control.

Article Snippet: Effects of CX3CL1 on differentiation and CX3CR1 expression of osteoclast precursors Osteoclast precursors (nonadherent mouse bone marrow cells) were cultured in -MEM supplemented with 10% FBS with or without 10 ng/ml murine recombinant chemokine domain CX3CL1 (10 nM; R&D Systems).

Techniques: Expressing, In Vitro, Derivative Assay, Cell Culture, Recombinant, Staining, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption.

doi: 10.4049/jimmunol.0803627

Figure Lengend Snippet: FIGURE 6. Anti-CX3CL1 mAb inhibits bone resorption in vivo. Neo- natal mice (n 3) were injected daily with control hamster IgG or hamster anti-CX3CL1 mAb (clone 5H8–4) for 5 days. After sacrifice, left femur sections were stained for TRAP and by toluidine blue. Three mice were analyzed for each group. A and B represent those treated with control IgG; C and D represent those treated with anti-CX3CL1 mAb. E, Epiphysis; OZ, osteogenic zone; GP, growth plate; AC, articular cartilage. TRAP-positive elongated cells on the bone surface are activated mature osteoclasts, which mediate bone resorption and invade and penetrate bone bits at the site of osteogenic zone. Original magnification: 4 (A, C); 40 (B, D). Repre- sentative results from experiments repeated at least three times are shown.

Article Snippet: Effects of CX3CL1 on differentiation and CX3CR1 expression of osteoclast precursors Osteoclast precursors (nonadherent mouse bone marrow cells) were cultured in -MEM supplemented with 10% FBS with or without 10 ng/ml murine recombinant chemokine domain CX3CL1 (10 nM; R&D Systems).

Techniques: In Vivo, Injection, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption.

doi: 10.4049/jimmunol.0803627

Figure Lengend Snippet: FIGURE 5. Anti-CX3CL1 mAb inhibits osteoclast differentiation in vitro. Osteoclast differentiation was induced in mouse bone marrow cells by coculture with primary osteoblasts in the presence of vitamin D3 for 6 days. Rat anti-CX3CL1 mAb (clone 126315) or control rat IgG was added to the culture medium at 20 g/ml. A, Picture of cocultured cells after TRAP-staining. Coculture was done in the presence of control rat IgG (a) or rat anti-CX3CL1 mAb (clone 126315; b). Asterisks indicate TRAP- positive osteoclasts. B, Effect of anti-CX3CL1 mAb on the number of TRAP-positive osteoclasts. TRAP-positive multinuclear cells containing three or more nuclei were counted as mature osteoclasts. C, Effect of anti- CX3CL1 mAb on the surface area of TRAP-positive osteoclasts. Surface areas of TRAP-positive osteoclasts were analyzed by image analysis soft- ware (NIH Image). Data represents the mean SD. , p 0.05. Repre- sentative results from experiments repeated at least three times are shown.

Article Snippet: Effects of CX3CL1 on differentiation and CX3CR1 expression of osteoclast precursors Osteoclast precursors (nonadherent mouse bone marrow cells) were cultured in -MEM supplemented with 10% FBS with or without 10 ng/ml murine recombinant chemokine domain CX3CL1 (10 nM; R&D Systems).

Techniques: In Vitro, Control, Staining

Journal: Molecular & Cellular Proteomics

Article Title: Proteomics Discovery of Metalloproteinase Substrates in the Cellular Context by iTRAQ™ Labeling Reveals a Diverse MMP-2 Substrate Degradome

doi: 10.1074/mcp.m600341-mcp200

Figure Lengend Snippet: FIG. 4. MMP-2 shedding of the chemokine domain of CX3CL1. A, schematic representation of full-length CX3CL1 using Swiss-Pdb- Viewer (Protein Data Bank code 1B2T). For mature CX3CL1 the po- sitions are shown of the iTRAQ-labeled tryptic peptides identified at 99% confidence in 3- and 48-h conditioned medium from Mmp2/

Article Snippet: Anti-FLAG M2 monoclonal antibody (F3165) was purchased from Sigma, anti-human MMP-2 monoclonal antibody (MAB13489) was purchased from Chemicon, human galectin-1 was purchased from Research Diagnostics, human heat shock protein-90 (HSP90 ) was purchased from Stressgen Bioreagents Corp., and recombinant extracellular domain (Met1–Arg337) of murine CX3CL1 (fractalkine) and anti-mouse CX3CL1 monoclonal antibody (MAB571) were purchased from R&D Systems.

Techniques: Multiplex sample analysis, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: CX3CL1 is elevated in chronic pain and impairs cognition via microglial CX3CR1. ( A , B ) ELISA quantification of CX3CL1 concentrations in the CSF (( A ), Control n = 34, Pain n = 18) and serum (( B ), Control n = 19, Pain n = 39) of clinical cohorts. ( C ) Receiver operating characteristic (ROC) curve evaluating the predictive accuracy of CSF CX3CL1 levels for chronic pain ( n = 34 for Control, n = 18 for Pain). The diagonal dashed line denotes the line of no discrimination. ( D ) MoCA scores reflecting the cognitive status of clinical patients (Control n = 15, Pain n = 23). ( E , F ) Pearson correlation analysis between CSF CX3CL1 concentrations and MoCA scores across the entire clinical cohort ( n = 30) ( E ) and specifically within the chronic pain group ( n = 18) ( F ). The solid line represents the linear regression line, with the shaded area indicating the 95% confidence interval. Sample sizes vary across cohorts ( A , B , D – F ) due to the non-interventional use of residual routine clinical specimens; all available unpaired data points were included without arbitrary exclusion. ( G – J ) Open field test (OFT) assessing ambulatory speed ( G ), total distance traveled ( H ), rearing frequency ( I ) and grooming frequency ( J ) in Control ( n = 8), CFA-induced chronic pain ( n = 7), CFA + PLX5622 (PLX, n = 8), and CFA + AZD8797 (AZD, n = 8) mouse groups. ( K , L ) Novel object recognition (NOR) test evaluating the recognition index ( K ) and total exploration time ( L ) across the four groups. ( M , N ) Escape latency to locate the hidden platform ( M ) and corresponding swimming speed ( N ) during the Morris water maze (MWM) training phase. ( O – Q ) Probe trial results of the MWM test, showing the time spent in the target quadrant ( O ), overall swimming speed ( P ), and representative swimming trajectories ( Q ). The red dashed circle indicates the former location of the target platform, which was removed during the probe trial. p -values were determined by unpaired two-tailed Student’s t -test ( A , B , D ), Pearson correlation analysis ( E , F ), one-way ANOVA followed by Tukey’s post hoc test ( G – L , O , P ), or two-way repeated-measures ANOVA with Tukey’s post hoc test ( M , N ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: Activation of microglial CX3CL1-CX3CR1 signaling induces neurotoxicity in co-cultured neurons. ( A ) Schematic illustration of the 0.4-μm Transwell system used for co-culturing pre-treated microglia with neuronal cells. ( B ) Western blot analysis of PSD95 and cleaved caspase-3 (c-Casp3) expression in PC12 cells co-cultured with BV2 microglia pre-treated with increasing concentrations of recombinant CX3CL1. ( C ) CCK-8 assay assessing the viability of PC12 cells following co-culture with CX3CL1-stimulated BV2 cells. ( D , E ) Validation of CX3CR1 knockdown efficiency in BV2 cells transfected with CX3CR1 siRNA via RT-qPCR ( D ) and Western blotting ( E ). ( F – I ) Western blot analysis of PSD95 and cleaved caspase-3 (c-Casp3) expression, alongside CCK-8 cell viability assays, in PC12 cells ( F , G ) and primary hippocampal neurons ( H , I ). Both neurons were co-cultured with BV2 cells subjected to CX3CR1 siRNA transfection and graded CX3CL1 pre-stimulation. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( C , D , G , I ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Recombinant, CCK-8 Assay, Co-Culture Assay, Biomarker Discovery, Knockdown, Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: CX3CL1-CX3CR1 signaling drives profound transcriptional reprogramming in microglia. ( A – C ) Volcano plots illustrating differentially expressed genes (DEGs) in BV2 cells across three comparisons: co-culture with PC12 cells versus monoculture ( A ), CX3CL1 pre-stimulation prior to co-culture versus vehicle co-culture ( B ), and CX3CR1 siRNA transfection plus CX3CL1 pre-stimulation versus CX3CL1 pre-stimulation alone ( C ). Significant DEGs were defined by ∣log2 (fold change) ∣ > 0.585 and adjusted p < 0.05. ( D – F ) Bubble plots displaying Gene Ontology (GO) enrichment analysis of the significant DEGs identified in the respective comparisons mentioned above. Vesicle-related and specific signal transduction terms are highlighted in bold blue.

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Co-Culture Assay, Transfection, Transduction

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: Microglial CX3CL1-CX3CR1 activation promotes small exosome secretion via Kifc2 upregulation. ( A ) Circos plot demonstrating the functional similarities among the significant DEGs identified in the three transcriptomic comparisons: co-culture with PC12 cells versus monoculture (brick red), CX3CL1 pre-stimulation prior to co-culture versus vehicle co-culture (indigo blue), and CX3CR1 siRNA transfection plus CX3CL1 pre-stimulation versus CX3CL1 pre-stimulation alone (forest green). Purple lines connect directly shared DEGs, whereas blue lines indicate extensive functional interconnectivity among non-overlapping DEGs. ( B ) Quadrant scatter plot comparing the fold changes of DEGs between the CX3CL1 pre-stimulated vs. control group, and the CX3CR1-depleted + CX3CL1 pre-stimulated vs. CX3CL1 pre-stimulated group. ( C ) RT-qPCR quantification of Kifc2 mRNA levels in BV2 cells under monoculture, co-culture, CX3CL1 pre-stimulated co-culture, and CX3CR1-depleted + CX3CL1 pre-stimulated co-culture conditions. ( D ) RT-qPCR validation of Kifc2 knockdown efficiency in BV2 cells transfected with Kifc2 siRNA. ( E – H ) Nanoparticle tracking analysis (NTA) of BV2-derived extracellular vesicles following CX3CL1 stimulation or transfection with Cx3cr1 or Kifc2 siRNAs, displaying the absolute concentration of small exosomes (<100 nm) ( E ), total EV concentration ( F ), relative proportion of small exosomes ( G ), and overall size distribution profile ( H ). ( I ) Western blot analysis of canonical positive exosome markers (CD63 and TSG101) and the negative control marker (Calnexin) in EV fractions and whole cell lysates and purified EV fractions under control or CX3CL1-stimulated conditions. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( C – G ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Activation Assay, Functional Assay, Co-Culture Assay, Transfection, Control, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Derivative Assay, Concentration Assay, Western Blot, Negative Control, Marker, Purification

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: The microglial CX3CL1-CX3CR1 signaling activates MAPK/NF-κB pathways and triggers the secretion of IL-17-enriched exosomes. ( A – C ) KEGG pathway enrichment analysis of the significant DEGs identified in three comparisons: co-culture with PC12 cells versus monoculture ( A ), CX3CL1 pre-stimulation prior to co-culture versus vehicle co-culture ( B ), and CX3CR1 siRNA transfection plus CX3CL1 pre-stimulation versus CX3CL1 pre-stimulation alone ( C ). Specific signal transduction terms are highlighted in bold blue. ( D ) ELISA quantification of IL-17 concentrations in EVs secreted by BV2 cells subjected to CX3CL1 stimulation, CX3CR1 siRNA, or Kifc2 siRNA transfection. ( E , F ) Western blot analysis of MAPK (p38, ERK, JNK) and NF-κB (p65) phosphorylation status in BV2 cells following stimulation with 50 ng/mL CX3CL1. ( G ) Predicted and experimental NF-κB binding loci within the Kifc2 locus, cross-referenced using ReMap ChIP-seq metadata and JASPAR motif analysis. ( H ) ChIP-qPCR assay validating p-p65 binding to the Kifc2 promoter. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( D , H ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Co-Culture Assay, Transfection, Transduction, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Binding Assay, ChIP-sequencing, ChIP-qPCR

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: The microglial CX3CL1-CX3CR1 signaling induces neurotoxicity by promoting IL-17-enriched exosome release via the p38 MAPK-NF-κB-KIFC2 axis. ( A , B ) Western blot analysis of p38 MAPK-NF-κB phosphorylation and KIFC2 expression in BV2 cells treated with CX3CL1, in the presence of the p38 inhibitor SB203580 (20 μM) or the NF-κB inhibitor MCCK1 (2 μM). ( C – F ) NTA characterization of EVs secreted by BV2 cells treated with CX3CL1 in combination with SB203580 or MCCK1, displaying representative size distribution profiles ( C ), absolute concentration of small exosomes (<100 nm) ( D ), total EV concentration ( E ), and the relative proportion of exosomes ( F ). ( G ) ELISA quantification of IL-17 concentrations in EVs secreted by BV2 cells following the indicated inhibitor treatments. ( H ) CCK-8 assay assessing the viability of PC12 cells co-cultured with BV2 cells pre-treated with CX3CL1, SB203580, or MCCK1. ( I ) Western blot analysis of PSD95 and cleaved caspase-3 (c-Casp3) expression in PC12 cells co-cultured with similarly pre-treated BV2 cells. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( D – H ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: Western Blot, Phospho-proteomics, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Aberrant CX3CL1-CX3CR1 Signaling Reprograms Microglial Exosome Secretion via KIFC2 to Drive Cognitive Impairment in Chronic Pain

doi: 10.3390/ijms27146304

Figure Lengend Snippet: In vivo validation of the microglial CX3CL1-CX3CR1-p38 MAPK-NF-κB-KIFC2 neurotoxic axis in the hippocampus. ( A , B ) Representative immunofluorescence images and corresponding quantification showing the expression of Iba1, p-NF-κB (p-p65) ( A ), and Kifc2 ( B ) in the hippocampal dentate gyrus of Control ( n = 8), CFA ( n = 7), CFA + PLX5622 (PLX, n = 8), and CFA + AZD8797 (AZD, n = 8) mice. For the representative images, low-magnification overviews are shown on the left, with white dashed boxes outlining the regions of interest that are displayed at higher magnification on the right. Scale bars = 100 μm. ( C ) A schematic model delineating the proposed mechanism: aberrant activation of the CX3CL1-CX3CR1-p38 MAPK-NF-κB-KIFC2 cascade in microglia drives secretome reprogramming, leading to the massive release of IL-17-enriched exosomes that subsequently precipitate neuronal injury. Red upward arrows indicate the up-regulation of cleaved caspase-3, while blue downward arrows indicate the down-regulation of PSD95. p -values were determined by one-way ANOVA followed by Tukey’s post hoc test ( A , B ).

Article Snippet: For pharmacological inhibition, BV2 cells were pre-incubated with either the p38 inhibitor SB203580 (HY-10256, MedChemExpress, 20 μM) or the NF-κB inhibitor MCCK1 (HY-D0162R, MedChemExpress, 2 μM) for 12 h, followed by stimulation with recombinant murine CX3CL1 (RP01393, ABclonal, Woburn, MA, USA) for 24 h. For gene silencing experiments, BV2 cells were transfected with small interfering RNAs (siRNAs) targeting Kifc2 or Cx3cr1 for 36 h, followed by a 24 h stimulation with recombinant CX3CL1.

Techniques: In Vivo, Biomarker Discovery, Immunofluorescence, Expressing, Control, Activation Assay

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Characterization and effects of morphine and/or HIV-1 Tat exposure on bound and soluble fractalkine, and CX 3 CR1 levels in mixed striatal neuron-glia cultures . Fractalkine immunofluorescence was readily co-localized in MAP2-positive neuron cell bodies and dendrites (A), while a majority of the CX 3 CR1 immunoreactive cells were CD11b-positive microglia (B); scale bar = 15 μm. Fractalkine derived from neuron-glial co-cultures presented a prominent band at approximately 95-kDa (C). This is slightly higher than the non-glycosylated, recombinant fractalkine (rFKN) control protein detected at 90-kDa; 0.1 μg and 0.025 μg fractalkine (FKN) were loaded onto the left-hand lanes. The relative abundance of either variant appeared to be unaffected by morphine (Morph) and/or Tat treatment at 24 h (C); n = 3 experiments. The amount of fractalkine released into the culture medium was unaffected following 2 h of morphine and/or Tat exposure (D); the limit of fractalkine detection by ELISA was 320 pg/ml (standard curve in D); n = 3 experiments. Unlike fractalkine, treatment with morphine and Tat together significantly reduced CX 3 CR1 levels (~47-kDa) at 24 h, but not 6 h or 12 h, compared to vehicle-treated controls (E) (* P < 0.02 vs. vehicle-treated controls; Kruskal-Wallis ANOVA and nonparametric post hoc tests), while CX 3 CR1 levels were unaffected by exposure to morphine or Tat alone (E); fractalkine and CX 3 CR1 levels were normalized to actin (~43-kDa bands in C,E) in immunoblots; n = 6 experiments.

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques: Immunofluorescence, Derivative Assay, Recombinant, Control, Variant Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Effects of fractalkine on morphine and/or HIV-1 Tat-induced toxicity was repeatedly tracked in individual striatal neurons (A-E) . A within-subjects design and computer-assisted, time-lapse tracking of individual neurons is used to compare the survival of the same neuron before and at 20 min intervals throughout treatments (A-E). Thriving neurons are evident in control (A; neurons 1-3) or morphine (Morph) + Tat-treated cultures co-administered fractalkine (B; neurons 1-4) at 48 h. By contrast, increased neurodegeneration and death was apparent with morphine plus Tat exposure (C). Neuron death is often preceded by the systematic degeneration of neurites and fragmentation of the cell body (Tat + morphine treatment; arrowheads, neuron 1), while death per se occurs precipitously; denoted here by fragmentation of the cell body and some loss of birefringence (arrow, neuron 1; 44 h and 48 h) (C). Some individual microglia that were tracked are encircled in red, orange, and yellow, respectively (arrowheads indicate prior movement); there was close association of microglia with the dying (1) and surviving (2) neuron (not all microglia in the fields are encircled) (C). Neuron death has been confirmed using other viability markers (see text). Many neurons remain viable despite morphine and Tat co-exposure (e.g., neuron 2); some surrounding cells, including astroglia, immature glial precursors and microglia, can display sporadic movement; scale bars = 20 μm. While Tat alone did not cause significant neurotoxicity relative to controls by 48 h [ , ]; there was a significant interaction when overall neuron losses were compared in Tat versus non-Tat vehicle-treated cultures ( § P < 0.03; Tat × time vs. Vehicle-control × time). Compared to controls or exposure to fractalkine alone, combined morphine and Tat (Morph+Tat) treatment increased neuronal death at 48 h (D) (* P < 0.05 vs. controls). Exogenous fractalkine (1 μg/ml) (red markers and connecting lines) prevented the accelerated neuronal death caused by combined Morph+Tat ( # P < 0.05 vs. Morph+Tat) (D). By contrast, CX 3 CR1 immunoblockade (blue markers and connecting lines) by itself caused significant neurotoxicity that was indistinguishable from the combined effects of Morph+Tat (E) (* P < 0.05 vs. controls). Moreover, when CX 3 CR1 blockade was combined with Morph+Tat, there were no additive neuron losses (E). The findings suggest a critical role for fractalkine-CX 3 CR1 signalling in potentiating the toxic effects of opioids in Tat-exposed neurons; data are the mean number of surviving neurons (compared to pretreatment numbers) ± SEM from n = 3-6 experiments; wild-type (WT) neurons and mixed glia; antibodies (Ab); scale bar = 20 μm.

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques: Control

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Studies with Cx3cr1 -/- mice: Fluorescent images and effects of Cx3cr1 -/- glia on fractalkine-mediated neuroprotection . Images of the striatum (A-C) and of mixed glial cultures (D) from Cx3cr1 -/- mouse striata. Microglia within the striatum of Cx3cr1 -/- ( Cx3cr1 GFP/GFP ) mice fluoresce green (A,B) and are Iba-1 immunoreactive (C); Hoechst counterstained nuclei (blue); scale bars = 50 μm (A) and 10 μm (B,C). Robust GFP expression is maintained when the cells are placed into culture (D). As can be appreciated from the Hoechst stain, the CX 3 CR1-expressing population is significant although the majority of the cells in the culture lack CX 3 CR1. The loss of CX 3 CR1 on glia has a dramatic effect on the ability of fractalkine to protect morphine and Tat-treated striatal neurons (E). Neurons from the striata of wild-type mice were plated onto mixed glia prepared from Cx3cr1 -/- mouse striata and followed over 48 h using our standard paradigm. In general, wild-type neurons co-cultured with Cx3cr1 -/- glia appeared to be less viable than with wild-type glia (upper panel; compare gray and black dotted lines); however, these groups were not compared statistically since the experiments were not run concurrently. The neuroprotective effects of fractalkine (CX 3 CL1; 1 μg/ml) are abolished when wild-type neurons are co-cultured with Cx3cr1 -/- mixed glia. Under these conditions, fractalkine no longer protects neurons from combined morphine and Tat exposure (E, lower panel; * P < 0.05 vs. vehicle- or fractalkine-treated controls). There were no differences in survival between controls ± fractalkine. Addition of fractalkine did not change the toxicity of morphine, Tat, or morphine + Tat treatments. There is, however, some possibility that fractalkine enhances baseline neuron survival in the Cx3cr1 -/- glial co-cultures. This was indicated by significant differences only at 48 h between Tat + fractalkine versus control + fractalkine survival ( § P < 0.05), even though survival did not differ when comparing vehicle + control vs. fractalkine + control or vehicle + Tat vs. fractalkine + Tat treatments. A similar inconsistency was observed only at 48 h with morphine treatments ( § P < 0.05).

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques: Expressing, Staining, Cell Culture, Control

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Effects of morphine and/or HIV-1 Tat exposure ± fractalkine on the percentage of microglia in mixed-glia cultures from striata of Cx3cr1 -/- ( Cx3cr1 GFP/GFP ) mice ( ‡ ).

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques: Mouse Assay, Control

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Effects of fractalkine on morphine and/or Tat-induced alterations in microglial motility (A-D) and nitrosative stress (E-F) . Morphine and Tat together (Morph+Tat) increased microglial movement in time-lapse images (representative cells circled in red) (A,B), while concurrent fractalkine exposure negated the effects of Morph+Tat (representative cells encircled in red) (B,C). When the vectorial trajectories of individual microglial movement are plotted each hour for 6 h, (i) the sporadic movements of individual cells and (ii) the ability of fractalkine to attenuate the overall motility are appreciated (B). Measurements confirmed that combined Morph+Tat increased microglia motility compared to vehicle, morphine, or Tat exposure alone (* P < 0.05). Addition of exogenous fractalkine (1 μg/ml) completely reversed the effect of Morph+Tat (D) ( # P < 0.05 vs. Morph+Tat alone), returning motility to baseline levels. The average movement of ~100 microglia in each treatment group was determined by measuring the distance from nuclear position at 0 h vs. 12 h per treatment in each experiment; values are the mean movement (μm/h) ± SEM from n = 4 experiments. Representative microglia that were tracked are encircled in red; the occasional macrophage/microglial interlopers that appeared and/or disappeared within the observation field and could not be tracked are circled in yellow; scale bar = 20 μm.

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Effects of fractalkine or immunoneutralizing anti-CX 3 CR1 antibodies on morphine and/or Tat-induced degeneration of MAP2-immunoreactive dendrites of striatal neurons at 72 h (A-E) . Compared to vehicle controls (Con) (A), morphine and Tat (Morph+Tat) (B) caused significant reductions in dendritic length (* P < 0.05 vs. vehicle controls) (E). Fractalkine prevented Morph+Tat-induced dendritic pruning ( # P < 0.05 vs. Morph+Tat) (D,E), but by itself had no effect (C,E). Data in E are mean dendrite length (μm) ± SEM from n = 4 experiments; scale bar = 20 μm.

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: Fractalkine/CX 3 CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

doi: 10.1186/1750-1326-6-78

Figure Lengend Snippet: Effects of fractalkine or immunoneutralizing anti-CX 3 CR1 antibodies on morphine and/or Tat-induced TNF-α production by mixed neuron-glial cultures at 12 h . Tat ± morphine (Morph) significantly increased TNF-α production by mixed neuronal-glial cultures from striatum (* P < 0.05 vs. vehicle-treated controls); Tat ± morphine-induced TNF-α production was unaffected by fractalkine or CX 3 CR1 immunoblockade, although there was a trend toward increased TNF-α release in control and morphine-treated cultures with CX 3 CR1 blockade.

Article Snippet: Preabsorbed fractalkine antibody controls consisted of rabbit anti-murine fractalkine antibody (R&D Systems) IgG preabsorbed with 25 μg/ml of the recombinant fractalkine peptide epitope used for antibody production for 1 h at 37°C, 5% CO 2 and in high humidity.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: CX3CL1 release during immunogenic apoptosis is associated with enhanced anti-tumour immunity

doi: 10.3389/fimmu.2024.1396349

Figure Lengend Snippet: CX3CL1 release is associated with immunogenic apoptosis. (A, D) Cell death measured by flow cytometry of MCA205 cells (A) and B16-F10 cells (D) treated with 2 μM or 8 μM MTX (MCA205 and B16-F10 respectively), 2.5 μM RSL3, or three cycles of F/T. Quantification was done by AnV and Sytox Blue staining. The values are the means ± SEM and represent three independent experiments. (B, E) Representative dot plots of the cell death measurement shown in (A, D) . (C, F) The concentration (pg/mL) of CX3CL1 measured in the supernatants of dying cells using Luminex xMAP technology. The values are the means ± SEM and represent three independent experiments. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparisons test: **p < 0.01, ****p < 0.0001. MTX, mitoxantrone; RSL3, RAS-lethal selective 3; F/T, freeze/thaw; AnV, Annexin-V; Sytox, Sytox Blue.

Article Snippet: MCA205 cancer cells were seeded at a density of 2 x 10 6 cells per flask and induced to undergo apoptosis with 2 μM MTX for 24 h. After incubation overnight, 2.5 x 10 5 dying cancer cells (non-immunogenic dose) were collected and mixed with different doses (0, 1, 10 or 100 ng) of recombinant murine CX3CL1 (R&D system, 472-FF/CF) in PBS at a volume of 200 μL per mouse.

Techniques: Flow Cytometry, Staining, Concentration Assay, Luminex

Journal: Frontiers in Immunology

Article Title: CX3CL1 release during immunogenic apoptosis is associated with enhanced anti-tumour immunity

doi: 10.3389/fimmu.2024.1396349

Figure Lengend Snippet: CX3CL1 reverts non-immunogenic apoptosis to ICD. (A) Schematic representation of the tumour prophylactic vaccination mouse model. On day 0, mice were vaccinated in the left flank with either 5 x 10 5 or 2.5 x 10 5 MTX-treated MCA205 cells. On day 7, the mice were challenged in the opposite flank with 10 5 viable cancer cells of the same type and tumour growth was monitored with a digital calliper. (B) Kaplan-Meier curve of the progression of tumour development over time. The reduction of dose of MTX-treated MCA205 cells from 5 x 10 5 cells to 2.5 x 10 5 cells significantly decreased tumour-free survival from 70% to 20%. The statistical differences were calculated by a log-rank (Mantel-Cox) test. Survival curves comparison: **p < 0.01. (C) Schematic representation of the tumour prophylactic vaccination mouse model. On day 0, mice were vaccinated in the left flank with either PBS, 2.5 x 10 5 MTX-treated MCA205 cells alone or 2.5 x 10 5 MTX-treated cells in combination with different doses of recombinant CX3CL1 (1 ng, 10 ng or 100 ng). On day 7, mice were challenged in the opposite flank with 10 5 viable cancer cells of the same type and afterwards tumour growth was followed with a digital calliper. (D) Kaplan-Meier curve of the progression of tumour development over time. The addition of 1 ng or 10 ng of rCX3CL1 to MTX-treated MCA205 cells significantly increased tumour-free survival from 10% (MCA205 MTX alone) to 50%. Interestingly, 100 ng of rCX3CL1 had no significant effect on tumour-free survival. The statistical differences were calculated by a log-rank (Mantel-Cox) test. Survival curves comparison: *p < 0.05, **p < 0.01, ***p < 0.001. ICD, immunogenic cell death; MTX, mitoxantrone; PBS, phosphate-buffered saline; rCX3CL1, recombinant CX3CL1.

Article Snippet: MCA205 cancer cells were seeded at a density of 2 x 10 6 cells per flask and induced to undergo apoptosis with 2 μM MTX for 24 h. After incubation overnight, 2.5 x 10 5 dying cancer cells (non-immunogenic dose) were collected and mixed with different doses (0, 1, 10 or 100 ng) of recombinant murine CX3CL1 (R&D system, 472-FF/CF) in PBS at a volume of 200 μL per mouse.

Techniques: Comparison, Recombinant, Saline