The decellularized pcECM was frozen in liquid nitrogen, crushed and lyophilized to a dry fine powder. The powder was solubilized (10 mg ml−1) in HCl (0.01 M) using sonication (1 min), followed by enzymatic digestion using pepsin (1 mg ml−1). The solution was then adjusted to pH = 5 with NaOH and kept cold (4 °C). To crosslink the solubilized pcECM, genipin (Sigma-Aldrich, 0.01 gr genipin/gr ECM) was added, and the samples were plated at 37 °C for 3 hrs. Following, PBS was added to prevent dehydration24 (link).
Genipin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China
Genipin is a natural compound derived from the fruit of the Gardenia jasminoides plant. It functions as a cross-linking agent, capable of forming stable covalent bonds with various biomolecules, including proteins, amino acids, and polysaccharides.
Automatically generated - may contain errors
Lab products found in correlation
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73 protocols using genipin
1
Decellularized pcECM Crosslinking Protocol
2
Genipin Crosslinking of Compressed Membranes
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Under sterile conditions, half of the compressed membranes created at 400 g, 1450 g and 3000 g were placed in six well culture plates and submerged in 4 mL of 1% genipin (Sigma Aldrich, St. Louis, MO, USA) for 48 h. After 48 h, membranes were washed twice with PBS to remove all unbound genipin. All uncrosslinked (UN-XL) and crosslinked (XL) membranes were stored at 4 °C in PBS solution until analysis.
3
Fabrication of Polyelectrolyte Multilayer Membranes
4
Preparation of Genipin Stock Solution
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Genipin (purity ≥ 98%) was obtained from Sigma–Aldrich (USA) and as a gift from Dr. Jeong from Duksung Women's University. Genipin was dissolved in dimethyl sulfoxide (DMSO) (Sigma–Aldrich), and a 200 mM stock solution was prepared. The solution was then filtered through a 0.22-μm filter (France) and stored at −20°C until use.
5
Development of Bronchial Tree Model
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NHBE cells and HUVECs were co-cultured to develop a bronchial tree46 (link). HUVECs were dispersed in growth factor-reduced matrigel (#356230; Corning, NY) with different concentrations of genipin (G4796; Millipore Sigma; MA, USA), with a cell density of 3.0 × 103 cells/μL of matrigel, then gently mixed with a pipette. Two hundred microliter of mixed matrigel was then injected on a ϕ 12-mm cover glass placed on a 12-well plate to sustain a sufficient matrigel height. Next, condensed NHBE (5.0 × 104 cells/μL) taken from the pellet of cells obtained after centrifugation was injected into the centre of the HUVEC-matrigel gel, and the sample was incubated at 37 °C for 25 min to facilitate gelation. Two millilitres of B + E medium was added to each well, and the medium was changed every day.
6
Radiolabeled Compound Synthesis
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Tritium gas was purchased from American Radiolabeled Chemicals, St Louis, MO. potassium [ 14 C]cyanide was purchased from Tjaden Biosciences, Burlington IA, Genipin was purchased from MilliporeSigma, St Louis, MO. Ketone 5 was synthesized by RTI Discovery Sciences and used as is. All remaining reagents were purchased from MilliporeSigma or Acros Organics, Pittsburgh, PA and were used as received. All 14 C steps were performed under an atmosphere of nitrogen.
7
Rat Oxygen-Induced Retinopathy Protocol
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The rat OIR model used in this study has been described previously.18 (link),19 (link) Within 6 hours of birth, newborn Sprague-Dawley pups (male and female) and dam were placed into an oxygen chamber (Oxycycler, Biospherix, NY, USA) with controlled oxygen concentrations that cycled between 50% and 10% every 24 hours. After 14 days, pups were moved into room air (RA). Each litter had 12 to 14 pups to maintain consistency in measured outcomes in the model. Experiments were repeated in four individual litters.
In a litter, pups were randomly assigned to two groups and treated with intraperitoneal injections of genipin (10 mg/kg, Sigma-Aldrich Corp., St. Louis, MO, USA) dissolved in dimethyl sulfoxide (DMSO) and PBS (1:500) or an equal volume of vehicle (DMSO+PBS) as control beginning at postnatal day 3 (p3) and repeated every other day until p13. For pups in the OIR model, injections were performed at the end of 50% oxygen cycles and before 10% oxygen cycles to avoid interventions during the 10% oxygen cycle.
In a litter, pups were randomly assigned to two groups and treated with intraperitoneal injections of genipin (10 mg/kg, Sigma-Aldrich Corp., St. Louis, MO, USA) dissolved in dimethyl sulfoxide (DMSO) and PBS (1:500) or an equal volume of vehicle (DMSO+PBS) as control beginning at postnatal day 3 (p3) and repeated every other day until p13. For pups in the OIR model, injections were performed at the end of 50% oxygen cycles and before 10% oxygen cycles to avoid interventions during the 10% oxygen cycle.
8
Reconstitution of Mitochondrial Complexes
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KCl, Na2SO4, 2-(N-morpholino)ethanesulfonic acid (MES), tris(hydroxymethyl)aminomethane (Tris), EGTA, hexane, hexadecane, cytochrome c (Cyt c), decylubiquinone, KCN, sucrose, 3-(N-morpholino)propanesulfonic acid (MOPS), bovine serum albumin (BSA), arachidonic acid (AA), genipin, geniposide, adenine triphosphate (ATP), ammonium phosphate (monobasic, NH4H2PO4), dimethyl sulfoxide (DMSO), sulfo-NHS-acetate (NHS), methyl-4-nitrobenzenesulfonate (MNBS), N-ethylmaleimide (NEM), and diammonium salt of malic acid were purchased from Sigma-Aldrich (Munich, Germany). EDTA, KH2PO4, NaN3, and acetonitrile were purchased from Merck (Darmstadt, Germany). Diphytanoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cardiolipin (CL) came from Avanti Polar Lipids (Alabaster, AL). Chloroform was obtained from Carl Roth (Karlsruhe, Germany).
9
Optimized miRNA Repression Assays
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For miRNA repression assays, 20 ng of Renilla luciferase (RL) reporter plasmids with 500 ng of firefly luciferase (FL) plasmid was cotransfected in a 24-well format. RL/FL values were measured after 48 h.
For target RNA or FH-Ucp2 overexpression experiments, 1 µg of RL reporter or FH-Ucp2 expression plasmid was transfected per well of a six-well plate. The siRNAs were transfected at a concentration of 50 pmol in the six-well format. Pre–miR122-mimic (Ambion, Thermo Scientific) was transfected at 10 pmol in a 12-well format with RNAiMAX (Life Technologies) according to the manufacturer’s protocol.
All plasmid transfections were carried out with Lipofectamine 2000 and siRNAs or mirVana hsa-miR-122 mimic (Life Technologies) with RNAiMAX (Life Technologies) according to the manufacturer’s instructions. All Dharmacon SMARTpool ON-TARGETplus used were purchased from GE Healthcare.
The indicated cell types were treated with genipin (100 µM; Sigma-Aldrich) after 4 h of administering Ld infection to a total of 24 h. FCCP (Clontech, 0.5 µM) and oligomycin (Clontech, 2.5 µM) treatments were done for the indicated time periods.
For target RNA or FH-Ucp2 overexpression experiments, 1 µg of RL reporter or FH-Ucp2 expression plasmid was transfected per well of a six-well plate. The siRNAs were transfected at a concentration of 50 pmol in the six-well format. Pre–miR122-mimic (Ambion, Thermo Scientific) was transfected at 10 pmol in a 12-well format with RNAiMAX (Life Technologies) according to the manufacturer’s protocol.
All plasmid transfections were carried out with Lipofectamine 2000 and siRNAs or mirVana hsa-miR-122 mimic (Life Technologies) with RNAiMAX (Life Technologies) according to the manufacturer’s instructions. All Dharmacon SMARTpool ON-TARGETplus used were purchased from GE Healthcare.
The indicated cell types were treated with genipin (100 µM; Sigma-Aldrich) after 4 h of administering Ld infection to a total of 24 h. FCCP (Clontech, 0.5 µM) and oligomycin (Clontech, 2.5 µM) treatments were done for the indicated time periods.
10
Mitochondrial Function Assay Protocol
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Luciferin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), pyruvate, palmitate, α-cyano-4-hydroxycinnamic acid (α-CCA), isoproterenol (ISO), blebbistatin, cyclosporin A (CsA), antimycin A, and genipin were from Sigma. Ethylene glycol tetraacetic acid (EGTA) was from Amresco. ABT-737 (ABT) was from Selleck Chemicals (Houston, Texas). Rotenone was from Calbiochem. MitoTEMPO was from Enzo Life Sciences. Mg2+-Green AM, Rhod-4 AM, BCECF AM, and mitoSOX were from Invitrogen (Eugene, Oregon). The tetrapeptide SS31 (D-arg-dmt-lys-phe-NH2) was synthesized as described (Zhao et al., 2004 (link)).
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