For the incorporation of cells into PRP gels, PRP aliquots (pool of 3 donors) were thawed. Cells were seeded in PRP gels at a density of 2.5 × 103 cells per μl gel. For time-lapse microscopy, a gel volume of 20 μl was seeded in μ-slides (μ-Slide Angiogenesis, Ibidi); for histology and gene expression analysis, 120 μl gels were prepared in 96-well plates. Monocultures of each cell type and cocultures were performed as follows: 100% MSCs (100 M), 100% HUVECs (100 H), 75% HUVECs–25% MSCs (75 HM), and 50% HUVECs–50% MSCs (50 HM). Cells were resuspended in PRP and gelation induced by addition of human thrombin (Tisseel, Baxter, final concentration 5 U/ml) and incubation at 37°C for 15–30 min. All gels were cultured under static conditions at 37°C and 5% CO2 in EGM-2 growth medium (Lonza) with 5 μM ε-aminocaproic acid (Sigma).
ε aminocaproic acid
Manufactured by Merck Group
Sourced in United States
ε-aminocaproic acid is a chemical compound used as a laboratory reagent. It functions as an inhibitor of the enzyme plasmin, which is involved in the breakdown of blood clots. The compound is commonly used in research and analytical applications where the control of plasmin activity is required.
Automatically generated - may contain errors
Lab products found in correlation
μ slide angiogenesis, by Ibidi (1 mentions)
Human thrombin, by Baxter (1 mentions)
Egm 2 growth medium, by Lonza (1 mentions)
Plasminogen, by Merck Group (1 mentions)
Decitabine, by Merck Group (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Hpaf 2, by American Type Culture Collection (1 mentions)
Panc10, by American Type Culture Collection (1 mentions)
Fibrillar collagen type 1, by Chrono-Log (1 mentions)
Af647 conjugated human fibrinogen, by Thermo Fisher Scientific (1 mentions)
Dade innovin lipidated tissue factor tf, by Siemens (1 mentions)
D phe pro arg cmk ppack, by Santa Cruz Biotechnology (1 mentions)
Sigmacote, by Merck Group (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Corn trypsin inhibitor cti, by Prolytix (1 mentions)
H gly pro arg pro oh acetate salt gprp, by Bachem (1 mentions)
Tranexamic acid, by Merck Group (1 mentions)
Spectramax id3 plate reader, by Molecular Devices (1 mentions)
Cbs0065, by Stago (1 mentions)
8 protocols using ε aminocaproic acid
1
Cell Incorporation into Platelet-Rich Plasma Gels
2
Pancreatic Cancer Cell Line Culture
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The Panc‐1 (CRL‐1469, male), Panc10.05 (CRL‐2547, male), and HPAF‐II (CRL‐1997, male) cell lines were purchased from the American Type Culture Collection (ATCC). The AsPC‐1 (female) and Bx‐PC3 (female) cell lines were a generous gift from Dr. David Hoskin (Dalhousie University, Halifax, Nova Scotia, Canada). All cell lines tested negative for mycoplasma. Panc‐1 cells were supplemented with Dulbecco's modified Eagle's media with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Hyclone). AsPC‐1 and BxPC‐3 cells were supplemented with Roswell Park Memorial Institute with 10% fetal bovine serum and 1% pencillin/streptomycin. All cells were maintained at 37 °C with 5% CO2. Zarnestra (Tipifarnib; Cat no. S1453, Selleckchem, Houston, TX, USA) and decitabine (Cat no. A3656, Sigma Aldrich, Oakville, ON, Canada) were reconstituted in DMSO. Doxycycline (Cat no. 631311, Clontech, Mountain View, CA, USA) was reconstituted in tissue‐culture grade water. Plasminogen (Cat no. 528180, Sigma Aldrich), S2251 (Via Diapharma, Cat no. 82033239, West Chester, OH, USA), ε‐aminocaproic acid (Cat no. A2504, Sigma Aldrich), and aprotinin (Cat no. 800277, Pentapharm, Dornacherstrasse, Switzerland) were reconstituted in PBS.
3
Multicolor Flow Cytometry of Platelet Activation
4
Antithrombotic Agents in Zebrafish
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Dabigatran etexilate, apixaban, and rivaroxaban were dissolved in DMSO and diluted in embryo water to final concentrations of 50, 100, and 250 µM, respectively. At 5 dpf larvae were treated for 24 hours prior to laser-mediated endothelial injury on day 6. For ε-aminocaproic acid (Sigma) treatment, zebrafish embryos were incubated in 100 mM at 1 dpf and the time to occlusion was evaluated at 3 dpf following laser-induced endothelial injury.
5
T cells Plasmin Activity Assay
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T cells were cultured in black-walled clear bottom plates (Corning; catalog no.: 3603) before incubation for 2 h with plasmin or plasminogen (10 nM) and either ε-aminocaproic acid (Sigma–Aldrich, catalog no.: A2504), tranexamic acid (Sigma–Aldrich, catalog no.: PHR1812), or vehicle as indicated. After three washes with prewarmed AIM-V, 10 nM of H-D-Val-Leu-Lys-AMC acetate (I1390, Bachem) with or without 1 nM tPA was added. The plate was then sealed, and fluorescence was measured every 15 min for 16 h on either an EnVison Multilabel plate reader (PerkinElmer) or a Spectramax ID3 plate reader (Molecular Devices).
6
Extracellular Vesicle Plasmin Generation
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The plasmin generation capacity of the EVs was assessed by isolating EVs and measuring their ability to initiate plasmin generation using a chromogenic assay [10 ,11 ]. In brief, 200 µL citrate-anticoagulated plasma was diluted (1:2) in PBS/BSA (phosphate buffer saline, 0.1% bovine serum albumin and 0.1% NaN3) and incubated for 1 h at room temperature under mild rotation with magnetic beads (M450 Dynabeads; InVitrogen/Dynal, Oslo, Norway) coated with specific antibodies (anti-CD15). Plasmin was generated by incubating plasminogen (4 μmol/L, Stago, Asnières, France) with the EVs in the presence of ε-aminocaproic acid (5 mmol/L, Sigma-Aldrich, Saint-Louis, MO) and was detected at 405 nm after incubation with a plasmin-selective chromogenic substrate ([methyl-malonyl]-hydroxypropylarginine-p-nitroaniline, CBS0065, Stago, 2 mmol/L). The kinetics were followed for 2 h at 37°C, and Vmax was measured in the linear part of each curve. The results were expressed in units of A405 nm × 10−3/minute.
7
Spectrophotometric Assay of Plasmin Activity
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Plasmin activity in skim milk was assayed using the spectrophotometric method of Korycka-Dahl et al. (1983) , with a slight modification. Plasmin activity, defined as the change in absorbance per minute, was determined by mixing 100 μL of skim milk with 1,400 μL of 50 mM Tris buffer in pH 7.4 at 37°C. The Tris buffer (Fisher Scientific) contained 110 mM NaCl (Fisher Scientific), 0.6 mM H-d-valyl-l-leucyl-l-lysinep-nitroanilide dihydrochloride (Sigma Chemical Co., St. Louis, MO), and 3.3 mM ε-amino caproic acid (Sigma Chemical Co.). The reaction mixture was incubated for 60 min at 37°C. The absorbance at 405 nm was measured at 30-min intervals using a ThermoSpectronic spectrophotometer (Fisher Scientific). The rate of pnitroanilide formation was computed from the linear part of the absorbance versus time curves. After a lag of about 30 min, the increase in absorbance was linear at least up to 3 h. A similar mixture without skim milk was used as a control.
8
Characterization of Therapeutic Monoclonal Antibodies
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2.1. Chemicals. Methanol (HPLC gradient grade) and acetic acid (100%) were obtained from VWR (Radnor, PA, USA). Ammonium acetate (>98%), sodium hydroxide, ε-Amino-caproic acid (>98%), hydroyxypropylcellulose Mw 100000 (HPC), carboxypeptidase B (CP-B) and formic acid (>98%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Water used to prepare buffers and sample solutions was obtained using an ELGA purelab UHQ PS water purification system (Bucks, UK). IdeS (Immunoglobulin-degrading enzyme of Streptococcus pyogenes) also named FabRICATOR and IgGZERO were purchased from Genovis (Lund, Sweden). Cetuximab (Erbitux®, Merck KGaA, Darmstadt, Germany) is a sterile, preservative-free solution for intravenous infusion containing 5 mg/mL of Cetuximab. Additionally to the mAbs it contains sodium chloride, glycine, polysorbate 80, citric acid monohydrate, sodium hydroxide and water for injections.
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