Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 1 Digital single-molecule nanopillar surface-enhanced Raman scattering (SERS) platform for parallel counting of four types of cytokines. SEM images of a pillar array side view, b nanoboxes, and c a single nanobox on the top of a pillar; d SERS spectra of nanoboxes conjugated with 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐ thiazoleacetic acid (MMTAA) Raman reporters; e workflow for multiplex counting of cytokines, including fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Multiplex Assay

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 4 Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques:

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 5 Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a, b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a. It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Incubation

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 6 Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony- stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Concentration Assay

Journal: Nature communications

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

doi: 10.1038/s41467-021-21431-w

Figure Lengend Snippet: Fig. 7 Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.

Article Snippet: FGF-2 (223-FB), G-CSF (214-CS), GM-CSF (215-GM), CX3CL1 (365-FR) cytokines; monoclonal anti-FGF-2 (MAB233), anti-G-CSF (MAB214), anti-GMCSF (MAB615), anti-CX3CL1 (MAB3652) antibodies; polyclonal anti-FGF-2 (AF233), anti-G-CSF (AF-214), anti-GM-CSF (AF-215), anti-CX3CL1 (AF-365) antibodies; and FGF-2 (DY233-05), G-CSF (DY214-05), GM-CSF (DY215-05), and CX3CL1 (DY365) ELISA kits were bought from R&D Systems.

Techniques: Concentration Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Fractalkine Activates a Signal Transduction Pathway Similar to P2Y 12 and Is Associated With Impaired Clopidogrel Responsiveness

doi: 10.1161/atvbaha.112.250720

Figure Lengend Snippet: Figure 2. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser157 (A) and Ser239 (B) in the presence of P2Y12 receptor inhibitor ticagrelor (Tica) was determined using flow cytometry. Samples were stimulated with prostaglandin E1 (PGE1, 1 μmol/L) and ADP (20 μmol/L) in the absence or presence of fractalkine (FKN, 1 μg/mL) and Tica (1 μg/mL). ADP significantly decreased PGE1-medi ated VASP phosphorylation, which was abrogated in the presence of Tica. In the presence of full P2Y12 blockade by Tica, FKN signifi cantly reduced PGE1-induced VASP phosphorylation. n=5–6; MFI indicates mean fluorescence intensity.

Article Snippet: Human serum FKN was determined using the Human CX3CL1/ Fractalkine Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Phospho-proteomics, Flow Cytometry, Fluorescence

Journal: Oncology reports

Article Title: Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

doi: 10.3892/or.2015.3941

Figure Lengend Snippet: Figure 2. Western blot analysis of Akt, ERK1/2 and STAT3 protein expression and time course of the induction of Akt, ERK1/2 and STAT3 activities by CX3CL1 in (A) seven multiple myeloma cell lines and in (B) RPMI-8226. These cells were treated with CX3CL1 (10 mM, 0-10 min), as indicated. Total Akt, ERK1/2 and STAT3 protein as well as their phosphorylated forms including Akt phosphorylated in serine (pSer473-Akt) and phosphorylated ERK1/2 (pThr-202/Tyr-

Article Snippet: Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&d Systems (Minneapolis, MN, uSA).

Techniques: Western Blot, Expressing

Journal: Oncology reports

Article Title: Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

doi: 10.3892/or.2015.3941

Figure Lengend Snippet: Figure 3. Anti-CX3CL1 antibody (10 µg/ml) was added after initiation of chemokine treatments. Akt and ERK1/2 phosphorylation in response to chemokine at 2 min was assessed in the absence (0) or presence of this antibody. Constitutively phosphorylated Akt and phosphorylated ERK1/2 in RPMI-8226 were inhibited even at low concentrations of anti-CX3CL1 antibody (10 µg/ml), whereas no changes were observed in their total protein expression. PCNA at the bottom of the panel served as a loading control. One representative experiment out of the three performed is shown.

Article Snippet: Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&d Systems (Minneapolis, MN, uSA).

Techniques: Phospho-proteomics, Expressing, Control

Journal: Oncology reports

Article Title: Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

doi: 10.3892/or.2015.3941

Figure Lengend Snippet: Figure 5. Induction of osteoclast differentiation by CX3CL1-pre-stimulating multiple myeloma. (A) The conditioned medium was prepared from RPMI-8226 cells stimulated with recombinant human CX3CL1 (10 nM) for 48 h. The RAW 264.7 cells were cultured with the 100 ng/ml of mouse recombinant soluble RANKL in the absence of 50% condition medium (a), in the presence of condition 50% medium (b), and in the presence of condition 50% medium and anti- CX3CL1 mAb. (B) After 4 days, TRAP-positive multinuclear cells containing three or more nuclei were counted as mature osteoclasts. Data represent the mean ± SD. *P<0.05. One representative experiment out of the three performed is shown.

Article Snippet: Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&d Systems (Minneapolis, MN, uSA).

Techniques: Recombinant, Cell Culture

Journal: Oncology reports

Article Title: Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

doi: 10.3892/or.2015.3941

Figure Lengend Snippet: Figure 4. CX3CL1 induces adhesion of multiple myeloma cells. RPMI-8226 was stimulated by recombinant human CX3CL1 (10 nM) for 5 min. The cells were added to wells that were pre-coated with fibronectin and VCAM-1 for 20 min and then washed. After stimulation, the RPMI-8226 cells were seeded and incubated for 20 min at 37˚C. The cells that adhered to the well were evaluated (pico green). Anti-CX3CL1 antibody (10 µg/ml) was used to ascertain the binding specificity. Data represent the mean ± SD. *P<0.05. One representative experiment out of the three performed is shown. VCAM-1, vascular cell adhesion molecule-1.

Article Snippet: Recombinant human CX3CL1 and anti-human CX3CL1 were purchased from R&d Systems (Minneapolis, MN, uSA).

Techniques: Recombinant, Incubation, Binding Assay

Journal: Cell death & disease

Article Title: A miR-125b/CSF1-CX3CL1/tumor-associated macrophage recruitment axis controls testicular germ cell tumor growth.

doi: 10.1038/s41419-018-1021-z

Figure Lengend Snippet: Fig. 5 Transcriptome analysis reveals gene regulation under miR-125b in NCCIT tumor cells. a Heatmap displaying 125 differentially expressed genes among miR-125b antagomir-, miR-125b agomir-, and NC-transfected NCCIT cells (Table S1). Differentially expressed genes were further clustered into two distinct clusters (cluster 1: miR-125b-negatively regulated genes, n = 18; cluster 2: miR-125b-positively regulated genes, n = 107). b Table of enriched biologic processes by KEGG analysis of differentially expressed genes. c Expression levels (FPKM values) of CSF1, CX3CL1, LTA, and AIMP1 that are suggested to exert macrophage recruitment activities. d, e Protein levels of CSF1 (d) and CX3CL1 (e) were detected in miR-125b knockdown, miR-125b overexpression, and NC NCCIT cells by Western blot analysis and normalized to GAPDH. Asterisks(**) indicates p < 0.01 by one- way ANOVA. Data were presented as the mean ± SEM

Article Snippet: The wild-type and mutant 3’ UTR of human CSF1 (~800 bp) and CX3CL1 (~1700 bp) were individually inserted into the pMirTarget (OriGene, CA, USA) using the EcoR1 and Xba1 sites.

Techniques: Transfection, Expressing, Knockdown, Over Expression, Western Blot

Journal: Cell death & disease

Article Title: A miR-125b/CSF1-CX3CL1/tumor-associated macrophage recruitment axis controls testicular germ cell tumor growth.

doi: 10.1038/s41419-018-1021-z

Figure Lengend Snippet: Fig. 6 CSF1 and CX3CL1 are served as direct targets of miR-125b in NCCIT tumor cells. a A schematic representation of the 3’ UTR luciferase reporter system and pMirTarget plasmid was shown. b, c 3’ UTRs of CSF1 (b) and CX3CL1 (c) harbor conserved seed match sequences (7-mer, CUCAGGG) for miR-125b recognition. d, e Luciferase assay in NCCIT cells was performed by co-transfection of Renilla, reporter plasmids carrying wild- type (WT) or mutant (MUT) 3’UTR, and miR-125b mimic or NC. Fold luciferase activity was displayed by the Firefly/Renilla ratio. Asterisks(**) indicated p < 0.01 and *** indicated p < 0.0001 by one-way ANOVA. Data were presented as the mean ± SEM

Article Snippet: The wild-type and mutant 3’ UTR of human CSF1 (~800 bp) and CX3CL1 (~1700 bp) were individually inserted into the pMirTarget (OriGene, CA, USA) using the EcoR1 and Xba1 sites.

Techniques: Luciferase, Plasmid Preparation, Cotransfection, Mutagenesis, Activity Assay

Journal: Cell death & disease

Article Title: A miR-125b/CSF1-CX3CL1/tumor-associated macrophage recruitment axis controls testicular germ cell tumor growth.

doi: 10.1038/s41419-018-1021-z

Figure Lengend Snippet: Fig. 7 miR-125b regulates CSF1 and CX3CL1 likely via a miRNA-mRNA network. a Heatmap displaying 47 differentially expressed genes among miR-125b antagomir-, miR-125b agomir-, and NC-transfected NCCIT tumor cells. Differentially expressed genes were further clustered into cluster 1 (genes positively regulated by miR-125b, n = 26) and cluster 2 (miR-125b-negatively regulated genes, n = 21). b Experimental procedure: overlap of predicted targets (by TargetScan) of differentially expressed miRNAs (revealed by miRNA-Seq) and differentially expressed mRNAs (revealed by mRNA-Seq). c Interaction network between miRNAs and target mRNAs. Red color indicated interaction association between miRNAs and CSF1. Green color highlighted network between miRNAs and CX3CL1

Article Snippet: The wild-type and mutant 3’ UTR of human CSF1 (~800 bp) and CX3CL1 (~1700 bp) were individually inserted into the pMirTarget (OriGene, CA, USA) using the EcoR1 and Xba1 sites.

Techniques: Transfection

Journal: Cell death & disease

Article Title: A miR-125b/CSF1-CX3CL1/tumor-associated macrophage recruitment axis controls testicular germ cell tumor growth.

doi: 10.1038/s41419-018-1021-z

Figure Lengend Snippet: Fig. 8 Possible mechanisms of miR-125b-mediated TAM recruitment and TGCT growth in vivo. Epigenetic modifications could account for the repressed miR-125b expression in TGCTs. miR- 125b overexpression significantly alleviated the tumor growth in vivo, likely due to the reduced abundance of TAMs. In mechanism, miR- 125b directly regulates the expression of tumor cell-derived chemokine CSF1 and CX3CL1, which are known to control the recruitment of TAMs to tumor sites

Article Snippet: The wild-type and mutant 3’ UTR of human CSF1 (~800 bp) and CX3CL1 (~1700 bp) were individually inserted into the pMirTarget (OriGene, CA, USA) using the EcoR1 and Xba1 sites.

Techniques: In Vivo, Expressing, Over Expression, Derivative Assay, Control

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Antihuman Fractalkine Reagents Used in this Study

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Control

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: Samples of Western lysates from WT CHO-K1 and CHO-K1 cells transfected with a human fractalkine expression vector 1 along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R&D Systems) (lanes 1–3) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd (lane 2, asterisk). These two bands are also detected by the chicken α-C-pep reagent (lane 5, asterisk). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant (lane 3, 85 to 90 kd), whereas the chicken α-C-pep does not (lane 6). Furthermore, the goat α-Fkn detects one larger (lane 2, 100 kd) and two smaller bands (lane 2, 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep (lane 5). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG1 control as a control for C. C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F. E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I. H: 3T3-Fkn stained with rabbit α-C-peptide. I: 3T3-Fkn stained with rabbit α-N-pep polyclonal reagent. 1 Note that although there is light nonspecific staining of the nucleus within the control sections (B, D, and F) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Membrane, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunohistochemistry, Staining, Control, Immunofluorescence

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R&D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 (A–D). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG1 mAb (Serotech), ii) goat α-Fkn polyclonal or 10% goat serum, iii) α-C-pep or rabbit IgG, iv) α-N-pep polyclonal 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Staining, Immunofluorescence, Labeling, Control, Incubation, Recombinant, Cell Culture, Reverse Transcription, Amplification, Expressing, Fluorescence