Journal: Sensors (Basel, Switzerland)

Article Title: Nanofluidic-Based Accumulation of Antigens for Miniaturized Immunoassay

doi: 10.3390/s20061615

Figure Lengend Snippet: Antigen-antibody recognition mechanism and biosensing experiments. ( a ) Positive test: target biotinylated IL10 antigens in solution recognize and bind to the probe antibodies anchored to the surface; fluorescent avidin binds to the biotin, and it is detected by fluorescence microscopy; ( b ) negative test: biotinylated not-matching antigens do not bind with the probe antibodies, and they are washed away, no fluorescence signal can be detected. ( c ) Epifluorescence microscopy images of the funnel after a positive test performed with a biotinylated IL10 concentration of 1.2 pg/mL (right) and after a negative test (left).

Article Snippet: In particular, we prepared solutions of matching antigens consisting of biotinylated recombinant human (rh) IL10 antigens (NF100 kit Fluorokine ® Biotinylated Human IL10, R&D system) diluted in PBS 1X at different concentrations (1.2 pg/mL, 12 pg/mL and 120 pg/mL) and of not-matching antigens, i.e., the negative control reagent provided by the NF100 kit (the soybean trypsin inhibitor biotinylated to the same degree as the cytokine).

Techniques: Avidin-Biotin Assay, Fluorescence, Microscopy, Epifluorescence Microscopy, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: Chondrocytes produce IL-7 in response to stimulation with fibronectin fragments. Human articular chondrocytes obtained from normal articular cartilage and cultured in serum-free media were treated overnight with 500 nmol/l of the 110 kDa fibronectin fragment (FN-f). Media was collected and analyzed for cytokine production using (a) an inflammation antibody array or (b) an IL-7 ELISA. Results are representative of three experiments for each result with different donor cells used in each experiment. The IL-7 spots on the array are shown in the red circles. (Other spots that were shown to change after fibronectin fragment stimulation included IL-6, soluble IL-6 receptor [sIL-6R], interferon-inducible protein [IP]-10, and monocyte chemotactic protein [MCP]-1.)

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Cell Culture, Ab Array, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: Effects of age and OA on chondrocyte production of IL-7. Media was collected 48 hours after changing to serum-free conditions in chondrocyte cultures established from (a) nonarthritic cartilage from 10 donors of different ages or from (b) cartilage from age-matched nonarthritic ( n = 7) and osteoarthritic cartilage ( n = 5). IL-7 was measured in the media using ELISA. The relationship of age to IL-7 levels was analyzed by Spearman correlation. The numbers in parentheses above the data points in panel a are the Collin's scores for the donor samples. OA, osteoarthritis.

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: Chondrocyte expression of IL-7 receptors. (a) Chondrocytes isolated from normal cartilage ( n = 1) were incubated with a fluorescently labeled recombinant IL-7 to demonstrate binding of IL-7 to the cell surface. Labeled cells were examined by flow cytometry. The peak that is shaded purple with the black line shows cells stained with IL-7, the peak with the pink line shows blocking antibody negative control, and the peak with the green line shows cells stained with the biotin negative control. (b) Chondrocytes isolated from normal cartilage were incubated with a fluorescently labeled recombinant IL-7 as above. Labeled cells were examined by confocal microscopy. IL-7 staining is shown in green. Top left is the green channel, top right is differential intermittent contrast, and bottom left is the merged image. Chondrocytes from eight different donors showed similar results. (c) Pooled RNA isolated from 10 different sets of cultured chondrocytes was subjected to reverse transcription and real-time PCR with an IL-7 receptor primer set. An amplification plot is shown to demonstrate positive signal. Amplified chondrocyte cDNA in triplicate is shown with the blue lines. Negative control with no reverse transcription of RNA before real-time PCR is shown with a red line. Negative control with no cDNA is shown with the black line.

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Expressing, Isolation, Incubation, Labeling, Recombinant, Binding Assay, Flow Cytometry, Staining, Blocking Assay, Negative Control, Confocal Microscopy, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Amplification

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: Chondrocytes respond to IL-7 stimulation with increased PYK2 phosphorylation and production of MMP-13. (a) Chondrocytes isolated from normal adult cartilage were stimulated with 10 ng/mL recombinant IL-7 and lysates were made at indicated time points for immunoblotting with an antibody to phosphorylated proline-rich tyrosine kinase (PYK)2 (Tyr402). The blot was then stripped and probed with total PYK2 antibody to confirm equal loading. (b) Densitometric scanning of the blot shown in panel a. (c) Medium was collected from serum-free chondrocyte cultures after overnight stimulation with 10 ng/ml recombinant IL-7 and examined for the presence of multiple matrix metalloproteinase (MMP) family members using an MMP antibody array. MMP-13 spots are shown in circles. (d,e) Media was collected from serum-free chondrocyte cultures after overnight stimulation with 10 ng/ml recombinant IL-7 or IL-1β, or the two together, and examined for the presence of MMP-13 using a commercially available ELISA. Results are the mean of seven experiments.

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Isolation, Recombinant, Western Blot, Ab Array, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: IL-7 causes proteoglycan release, but not nitric oxide production, in cartilage explants. Cartilage explants were stimulated for 72 hours with 10 ng/ml recombinant human IL-7 before media collection. (a) Medium was analyed for sulfated glycosaminoglycan (sGAG) using the dimethylmethylene blue assay and normalized for the wet weight of the tissue. (b) Total nitrite was measured in the media as a marker for nitric oxide production using commercially available colorimetric nitrate/nitrite assay kit. Results represent four experiments.

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Recombinant, Dimethylmethylene Blue Assay, Marker, Nitration

Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13

doi: 10.1186/ar2376

Figure Lengend Snippet: Role for IL-1 and IL-6 in stimulation of IL-7 production by chondrocytes. (a) Chondrocytes were pretreated with either an IL-6 neutralizing antibody or the IL-1 receptor antagonist, or the combination of the two inhibitors, and then subsequently stimulated with fibronectin fragment. After overnight stimulation media samples were collected and used for an inflammation antibody array. IL-7 spots are shown in red circles. (b) Chondrocytes were stimulated with either IL-1β (10 ng/ml) or IL-6/soluble IL-6 receptor (10 ng/ml and 20 ng/ml) or the combination of cytokines. Medium was collected and subsequently analyzed with an IL-7 ELISA.

Article Snippet: In both instances, cells were stained with fluorescently labeled IL-7 using the Human IL-7 Biotinylated Fluorokine Kit (R&D Systems), in accordance with the manufacturer's instructions but with slight modifications.

Techniques: Ab Array, Enzyme-linked Immunosorbent Assay

Journal: Developmental Cell

Article Title: Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus

doi: 10.1016/j.devcel.2023.08.017

Figure Lengend Snippet:

Article Snippet: HumanKine® recombinant human IL-7 protein , Proteintech , cat# HZ-1281.

Techniques: Recombinant, Modification, Selection, RNA Extraction, Polymer, Cell Culture, Derivative Assay, Software