Naringenin (C15H12O5) (4′,5,7-trihydroxyflavanone, molecular weight of 272.26, 95%purity, CAS number: 67604-48-2) was purchased from Glentham Life Sciences Ltd., Edinburgh, UK. The Amphorae coffeaeformis powder was supplied by members of Algal Biotechnology Unit (National Research Centre, Dokki, Giza, Egypt). Malondialdehyde (MDA) and reduced glutathione (GSH) assaying commercial diagnostic kits were purchased from the Biodiagnostic Company for Research Kits in Egypt. ELISA kits for rat disintegrin and metalloproteinase with thrombospondin 5 repeats (ADAM TS-5) (catalog number: SEK205Ra), matrix metalloproteinase-3 (MMP-3) (catalog number: LS-F5516.) and rat tissue inhibitor of metalloproteinase-3 (TIMP-3) (catalog number: RK03988) were provided by R&D System, Minneapolis, MN, USA. Indomethacin, monosodium iodoacetate, and all other compounds with high analytical grades were supplied by Sigma Chemical Company (St. Louis, MO, USA).
Timp 3
Manufactured by R&D Systems
Sourced in United States
TIMP-3 is a tissue inhibitor of metalloproteinases, a family of proteins that regulate the activity of matrix metalloproteinases. TIMP-3 plays a role in the control of extracellular matrix degradation.
Automatically generated - may contain errors
Lab products found in correlation
Timp 2, by R&D Systems (4 mentions)
Timp4, by R&D Systems (4 mentions)
Timp 1, by R&D Systems (4 mentions)
Gm6001, by Merck Group (2 mentions)
Tapi 2, by Santa Cruz Biotechnology (2 mentions)
Tapi 1, by Santa Cruz Biotechnology (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Monosodium iodoacetate, by Merck Group (1 mentions)
Adamts 5, by R&D Systems (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Mmp 3, by R&D Systems (1 mentions)
Recombinant timp1, by R&D Systems (1 mentions)
Phorbal 12 myristate 13 acetate, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Ns 398, by Cayman Chemical (1 mentions)
Sc 514, by Merck Group (1 mentions)
Ns 398, by Merck Group (1 mentions)
Sc 514, by Cayman Chemical (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
9 protocols using timp 3
1
Naringenin-based Osteoarthritis Treatment
2
CD200 and CD200R Antibodies in Immunoassays
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Phorbal 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma-Aldrich. PMA was reconstituted to 10mg/ml stocks in DMSO and was further diluted to a working concentration of 40ng/μl in AIMV medium. Imiquimod, a TLR7 agonist, was purchased from LKT Laboratories (St Paul, MN) and reconstituted to 1mg/ml in DMSO. Recombinant TIMP1, TIMP2, TIMP3, and TIMP4 were purchased from R&D Systems and were reconstituted to working concentrations in AIMV medium. The protease inhibitors GM6001 and TAPI-0 were purchased from Calbiochem and reconstituted to 10mM and 1mg/ml stock, respectively, in DMSO.
The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit serum against the extracellular region of CD200 (CD200v+c), were described previously [2 (link)]. A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was generated by immunization of rabbits with a fusion protein containing the extracellular region of human CD200R1 with a his-tag at the N-terminal.
Antibodies against CD19 and CD62L used in FACS analyses were purchased from Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane marker for loading controls in Western blots, was purchased from Abcam.
The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit serum against the extracellular region of CD200 (CD200v+c), were described previously [2 (link)]. A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was generated by immunization of rabbits with a fusion protein containing the extracellular region of human CD200R1 with a his-tag at the N-terminal.
Antibodies against CD19 and CD62L used in FACS analyses were purchased from Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane marker for loading controls in Western blots, was purchased from Abcam.
3
Screening Candidate Therapeutics in iPSC-OA Model
4
Quantifying TGF-beta Signaling Pathway
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We used the following materials and kits: recombinant human TIMP3 (R&D Systems, Wiesbaden, Germany) and recombinant human TGF-β1 and TGF-β2 (Promokine, Heidelberg Germany), protease inhibitor cocktail, Fluoroprofile protein quantification kit (Merck/Sigma Aldrich, Darmstadt, Germany), siRNAs (Thermo Fisher/Invitrogen, Carlsbad, CA, USA) and GM6001 (broad-spectrum inhibitor of MMPs; Sigma Aldrich, St. Louis, Missouri, USA), TGF-β1 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no MB100B), TGF-β2 (range 31.2–2000 pg/mL, sensitivity 15.6 pg/mL, cat-no DY302), and TIMP3 (range 62.5–4000 pg/mL, cat-no DY973) ELISAs (all from R&D Systems, Wiesbaden, Germany), TIMP1 (range 0.156–10 ng/mL, sensitivity 0.05 ng/mL, cat-no MBS263032), TIMP2 (range 78–5000pg/mL, sensitivity 39 pg/mL, cat-no MBS2880823), rat TβRIII/sBG ELISA (range 0.156–10 ng/mL, sensitivity 0.086 ng/mL; cat-no MBS289506, all from MyBioSource, San Diego, CA, USA), rat P-Smad3 ELISA (range 10–500 µg/mL, sensitivity 10 µg/mL; cat-no ab186038, Abcam, Cambridge, UK) and P-Smad2 ELISA (10–1000 µg/mL; Cell Signaling, cat-no 7384c, Frankfurt, Germany).
5
Assaying TIMP-3 Effects on Cell Proliferation
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The proliferation of SMCs was indirectly assayed using Cell Counting Kit-8 (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Rockville, MD, USA), according to the manufacturer's protocol. Briefly, cells were seeded at a density of 3×103 cells/100 µl/well in 96-well plates with 3, 10, 30, or 100 ng/ml of TIMP-3 (R&D Systems, Inc., Minneapolis, MN, USA) (4 wells/group) for 24 h at 37°C in a humidified 5% CO2 incubator. Cells cultured without TIMP-3 were used as the control group. Subsequently, 10 µl CCK-8 solution was added to each well, and the plates were incubated at 37°C for 3 h. The optical density of the wells was measured at 450 nm, using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
6
Multiplex ELISA Profiling of Cytokines
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A qualitative commercial enzyme-linked immunosorbent assay test (Multi-analyte ELISArray kit (SABiosciences - QIAGEN, Maryland, USA) has been used to simultaneously profile the level of multiple cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFNγ, TNF-α, GM-CSF) in cell culture supernatants.
A cutoff of twice the absorbance value (read at 450 nm) of the negative control for every cytokine (A450) was used and the results were reported as positive (A450 ≥ specific cutoff) or negative (A450 < specific cutoff).
Protein concentration of detectable cytokines IL-6, IL-8, MMP-3, MMP-13 (Boster Biological Technology, Fremont, CA, USA), TIMP-3 and TIMP-4 (R&D Systems, Minneapolis, MN, USA) was then determined in cell-free supernatants using quantitative commercially available ELISA kits.
A cutoff of twice the absorbance value (read at 450 nm) of the negative control for every cytokine (A450) was used and the results were reported as positive (A450 ≥ specific cutoff) or negative (A450 < specific cutoff).
Protein concentration of detectable cytokines IL-6, IL-8, MMP-3, MMP-13 (Boster Biological Technology, Fremont, CA, USA), TIMP-3 and TIMP-4 (R&D Systems, Minneapolis, MN, USA) was then determined in cell-free supernatants using quantitative commercially available ELISA kits.
7
Metalloprotease Inhibitor Evaluation Protocol
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Metalloprotease inhibitors, including BB94 (batimastat), TAPI-1 and TAPI-2 (Santa Cruz Biotechnology), and TIMP1, TIMP2, TIMP3, and TIMP4 (R&D Systems), were added at the time of GPR37L1 induction (or 5 h after transfection in the case of U87 MG cells) or 24 h after induction for TX14A experiments, and cells were then incubated for 24 h before lysis. For MG132 proteasome inhibition, cells were induced with doxycycline overnight and then treated with MG132 for 6 h before harvest. Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 was performed as per the protocol provided by R&D Systems.
8
Quantification of Nasal Tissue Proteins
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Tissue homogenates were prepared as previously described.21 Briefly, frozen nasal tissues were weighed and homogenized with an automated homogenizer (TissueLyser LT; Qiagen) for 2 min. The homogenates were then dissolved in 0.9% NaCl (1 ml of 0.9% NaCl per 0.1 g of tissue) with 1% protease inhibitor cocktail (Sigma‐Aldrich, St Louis, Mo) and centrifuged to collect the supernatants.
The prepared tissue homogenates were assayed for tenascin C (US Biological), periostin, MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐12, MMP‐13 and TIMP‐1, TIMP‐2, TIMP‐3, and TIMP‐4 (R & D Systems) by commercially available kits.
The prepared tissue homogenates were assayed for tenascin C (US Biological), periostin, MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐12, MMP‐13 and TIMP‐1, TIMP‐2, TIMP‐3, and TIMP‐4 (R & D Systems) by commercially available kits.
9
Metalloprotease Inhibitor Impacts on GPR37L1
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Metalloprotease inhibitors, including BB94 (batimastat), TAPI-1 and TAPI-2 (Santa Cruz Biotechnology), and TIMP1, TIMP2, TIMP3, and TIMP4 (R&D Systems), were added at the time of GPR37L1 induction (or 5 hours after transfection in the case of U87 MG cells) or 24 hours after induction for TX14A experiments, and cells were then incubated for 24 hours before lysis. For MG132 proteasome inhibition, cells were induced with doxycycline overnight and then treated with MG132 for 6 hours before harvest.
Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH 2 was performed as per the protocol provided by R&D Systems.
Verification of TIMP activity in vitro using activated MMP-2 and the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH 2 was performed as per the protocol provided by R&D Systems.
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