ECM-mimetic hydrogels (50 μl) were generated from a HEPES solution (20 mM, 150 nM NaCl, pH 7.4) containing 8 mg/ml human fibrinogen (Enzyme Research Laboratories), 1 mg/ml human plasma fibronectin (Sigma), 500 μg/ml human vitronectin (Peprotech), 50 ug/ml human tenascin C (R&D Systems), 50 μg/ml heparan sulfate (Sigma) and 500 ng/ml of PDGF-BB or IL-1Ra variants. Matrices were polymerised in Ultra-Low Cluster 96-well plate (Corning) at 37 °C for 2 h with 10 U/ml bovine thrombin (Sigma) and 5 mM CaCl2. Then, matrices were transferred to Ultra-Low Cluster 24-well plate (Corning) containing 500 μl of buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, pH 7.4). Control wells that served as 100% released control contained only PDGF-BB and IL-1Ra variants in 500 μl of the buffer. Every 24 h, buffers were removed from wells and kept at −20 °C. Wells were replenished with fresh release buffer. For the 100% release control well, 20 μl of buffer was taken out every day and stored at −20 °C. After 7 days, the cumulative release of PDGF-BB and IL-1Ra variants was quantified by ELISA using the 100% released control as reference (PDGF-BB DuoSet, IL-1Ra/IL-1F3 DuoSet; R&D Systems). For release assays with plasmin, the same method was used except that the release buffer contained 100 μU/ml of plasmin (Roche).
Plasmin
Manufactured by Roche
Sourced in Australia
Plasmin is a laboratory-grade enzyme that can break down fibrin, the main component of blood clots. It is commonly used in research settings to study coagulation and fibrinolysis processes.
Automatically generated - may contain errors
Lab products found in correlation
Ultra low cluster 24 well plate, by Corning (3 mentions)
Human fibrinogen, by Enzyme Research (3 mentions)
Fibrinogen, by Enzyme Research (3 mentions)
Bovine thrombin, by Merck Group (2 mentions)
Heparan sulfate, by Merck Group (1 mentions)
Human plasma fibronectin, by Merck Group (1 mentions)
Human vitronectin, by Thermo Fisher Scientific (1 mentions)
Human tenascin c, by R&D Systems (1 mentions)
Ultra low cluster 96 well plate, by Corning (1 mentions)
Human vegf duoset elisa, by R&D Systems (1 mentions)
96 well plate, by Corning (1 mentions)
Ultracel 30k, by Merck Group (1 mentions)
Vitronectin, by Promega (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Anti endomucin antibody, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Transferrin, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Human thrombin, by Merck Group (1 mentions)
9 protocols using plasmin
1
ECM-Mimetic Hydrogel for Growth Factor Release
2
ECM-Mimetic Hydrogel Release Kinetics
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ECM-mimetic hydrogels (50 μl) were generated as previously described16 (link). Briefly, the matrices were generated from HEPES solution (20 mM, 150 nM NaCl, pH 7.4) containing 8 mg/ml human fibrinogen, 1 mg/ml fibronectin, 500 μg/ml human vitronectin, 50 ug/ml tenascin C, 50 μg/ml heparan sulfate and 500 ng/ml of PDGF-BB or IL-1Ra variants. Matrices were polymerised in a 96-well plate (Corning) at 37 °C for 2 h with 10 U/ml bovine thrombin (Sigma) and 5 mM CaCl2. Then, matrices were transferred to Ultra Low Cluster 24-well plate (Corning) containing 500 μl of release buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, pH 7.4). Control wells that served as 100% released control contained only PDGF-BB and IL-1Ra variants in 500 μl of buffer. Every day, buffers were removed from wells and kept at -20 °C. Wells were replenished with fresh release buffer. For the 100% release control well, 20 μl of buffer was taken out every day and stored at −20 °C. After 7 days, the cumulative release of PDGF-BB and IL-1Ra variants was quantified by ELISA using the 100% released control as reference (PDGF-BB DuoSet, IL-1Ra/IL-1F3 DuoSet; R&D Systems). For release assays with plasmin, the same method was used but the release buffer contained 100 μU/ml of plasmin (Roche).
3
Plasmin-Mediated Cleavage of GST-AREG
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GST or GST-AREG26-38 (50 μg) were incubated with plasmin (Roche) at increasing concentration in PBS for 1 h (total volume of 250 μl). The reaction was stopped by boiling the samples and cleavage was analysed by SDS-PAGE.
4
VEGF-A Release from Fibrin Gels
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Fibrin gels (70 μL; n = 4 gels per group) were made of 10 mg/mL fibrinogen, 2 U/mL thrombin, 4 U/mL Factor XIIIa, and 5 mM CaCl2 in HEPES buffer, in which 1 μM of α2PI and 200 ng VEGF-A-PlGF-2123–144 and PDGF-BB-PlGF-2123–144 were added, were incubated for 1 h at 37 °C with 5% CO2. Then, the fibrin gels were transferred into a 24-well cell culture plates and incubated in 1 ml release buffer (Tris 20 mM, NaCl 150 mM, 0.1% BSA, Pen/Strep, pH 7.4) containing 2.5 nM plasmin (Roche, #10602361001). The plate was kept at 37 °C with 5% CO2 until the gels were fully degraded. The plasmin-containing buffer was daily replaced, collected and stored at −20 °C. The daily release of VEGF-A was quantified by ELISA (Human VEGF DuoSet ELISA, R&D systems) as instructed by the manufacturer and normalized to the total released amount.
5
Lung and Heart Extracellular Matrix Extraction
6
Immunohistochemistry of IGFBP-2 in Cancer
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FreeStyle 293-F (FS293F) cells and the mammalian expression vector pcDNA3.1 were from Invitrogen (Mt Waverley, VIC, Australia). Anti-IGFBP-2 polyclonal antibody was generated in-house and anti-rabbit HRP antibody was from Pierce (Scoresby, VIC, Australia). Plasmin, MMP-1 and -7 were from Roche (Dee Why, NSW, Australia), Merck Millipore and Chemicon Millipore (Kilsyth, VIC, Australia), respectively. Heparin salt and fibronectin were purchased from Sigma (Castle Hill, NSW, Australia) and vitronectin was from Promega (Madison, WI, USA). Antibodies used in immunohistochemistry were anti-endomucin antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and anti-rabbit Dylight649 antibody (Jackson Immuno Research, West-Grove, PA, USA). T84 (CCL-248) and SW480 (CCL-228) colon cancer, DU145 (CRL-2698) and LnCaP (CRL-1740) prostate cancer and the MCF-7 (HTB-22) breast cancer cell lines were cultured as recommended by the ATCC at 37 °C in 5% CO2. ITS (insulin 10 mg ml−1, transferrin 5.5 mg ml−1, sodium selenite 5 μg l−1, Sigma) was a supplement for serum-free cell cultures. Reagents for immunohistochemistry included Histolene from Fronine Laboratory Supplies and ProLong Gold antifade reagent from Life Technologies (Mulgrave, VIC, Australia).
7
Fibrinolysis Assay of Mouse Plasma
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Immediately after euthanasia of C57BL/6 J mice, blood was withdrawn by cardiac puncture. The whole blood was mixed with sodium citrate (100 mM, pH 7.4–7.8) at 9:1 ratio to prevent coagulation, and centrifuged at 1500 × g for 10 min. The plasma supernatant was collected and froze at −80 °C until use. Fluorescent fibrinogen (0.2 mg/mL final, about 10% w/w) was mixed to mouse plasma to enable visualization of gel degradation by fluorescent imaging. Recombinant α2PI (5 μM) was added in the fibrinogen mix. To induce clotting, CaCl2 in HEPES buffer was added to the fluorescent plasma mix to a 25 mM final concentration. Plasma clots were incubated for 1 h at 37 °C with 5% CO2 to ensure complete polymerization. The plasma gels were then transferred into a 24-well cell culture plate, 1 gel per well, and incubated in 1 ml release buffer (Tris 20 mM, NaCl 150 mM, 0.1% BSA, Pen/Strep, pH 7.4) containing 25 nM plasmin (Roche). The plate was kept at 37 °C with 5% CO2 until all the gels were fully degraded. The plasmin-containing buffer was daily refreshed, and the imaging was performed as previously described.
8
Controlled Release of P28 Peptide
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The SIS/PLA/P28/PlGF-2123-144* scaffolds were transferred to 2 mL sterile PBS solution with or without 4 mg mL−1 of collagenase type I (Sigma-Aldrich) or 100 μU of plasmin (Roche) at 37 °C for 21 days. The SIS/PLA/P28 scaffolds placed in pure PBS solution served as a control group. The eluate of the P28 peptide was detected at 12 h and 1, 2, 3, 5, 7, 10, 12, 14, 18, and 21 days after incubation. The supernatant was completely removed and replaced with fresh enzymatic solution at each time interval. The amount of P28 peptide in the collected supernatant was detected by HPLC, as previously described.33
9
Quantification of Growth Factor and IL-1Ra Release from Fibrin Matrices
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Fibrin matrices were generated with fibrinogen (8 mg/ml; Enzyme Research Laboratories), human thrombin (2 U/ml; Sigma-Aldrich), 5 mM CaCl2, and growth factors (500 ng/ml) or IL-1Ra variants. Fibrin matrices were polymerized at 37°C for 1 hour and transferred to an Ultra-Low Cluster 24-well plate (Corning) containing 500 μl of buffer [20 mM tris-HCl, 150 mM NaCl, and 0.1% BSA (pH 7.4)]. Control wells that served as 100% released control contained only the growth factor and IL-1Ra variants in 500 μl of buffer. Every 24 hours, buffers were removed, kept at −20°C, and replaced with fresh buffer. For the 100% release control well, 20 μl of buffer was taken out every day and stored at −20°C. After 7 days, the cumulative release of growth factor and IL-1Ra variants was quantified by ELISA using the 100% released control as reference (BMP-2 DuoSet, PDGF-BB DuoSet, and IL-1Ra/IL-1F3 DuoSet; R&D Systems). For release assay with matrix metalloproteinases, the same method was used, except that the release buffer contained plasmin (100 μU/ml; Roche).
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