After obtaining informed consent from pregnant mothers, we isolated, expand, and preconditioned human WJ-derived MSCs with thrombin (2 U/mL; Sigma Aldrich, Steinheim, Germany) at Good Manufacturing Practice (GMP) facility of the Samsung Stem Cells and Regenerative Medicine Institute following the guidelines for GMP, as described previously [13 (link)]. Briefly, the MSCs characteristics were confirmed using flow-cytometric analysis for cell surface markers (CD73, CD90, CD105, CD166, CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19) and in vitro differentiation assays for osteogenesis, adipogenesis, and chondrogenesis as described [24 (link)]. After reaching 90% confluence, we preconditioned WJ-derived MSCs with thrombin in a culture medium (α-MEM; Gibco, Life Technologies, Carlsbad, CA, USA) for 3 h. Control naïve MSCs were prepared in the same manner except for thrombin treatment.
Thrombin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Thrombin is a serine protease that plays a crucial role in the blood coagulation cascade. It is an enzyme that catalyzes the conversion of fibrinogen to fibrin, which is the primary component of blood clots. Thrombin is a key factor in the hemostatic process, responsible for regulating blood clotting and maintaining vascular integrity.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by Merck Group (4 mentions)
Glutathione resin, by GenScript (2 mentions)
Fibrinogen, by Merck Group (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Fibrinogen, by Thermo Fisher Scientific (1 mentions)
Star124p, by Bio-Rad (1 mentions)
Star207p, by Bio-Rad (1 mentions)
Vitrocol, by Advanced BioMatrix (1 mentions)
Human fibrinogen, by Merck Group (1 mentions)
Convulxin, by Santa Cruz Biotechnology (1 mentions)
Medium 199, by Lonza (1 mentions)
A23187, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Papain, by Thermo Fisher Scientific (1 mentions)
His60 ni superflow resin, by Takara Bio (1 mentions)
Rprotein a sepharose, by GE Healthcare (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Tissue culture plates, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
12 protocols using thrombin
1
Thrombin-Preconditioned Human WJ-MSCs Protocol
2
Bacterial Protein Purification and Activation
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C2C was prepared as described previously19 (link). Briefly, plasmid encoded expression constructs for C2I and C2II-C1 were expressed in E. coli BL21. Cell lines were grown in LB medium, with ampicillin (100 µg/mL) at 37 °C, and induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM IPTG. A french pressure cell was used to lyse cell paste with 10,000 psi pressure. Glutathione resin (Genscript) was used for affinity purification. GST fusion tags were removed using thrombin (Thermo Fisher). C2II-C1 was further activated using trypsin by incubation at 37 °C for 30 min at a 1:5 enzyme to substrate ratio as previously described63 (link).
3
Fibrin Scaffold for Endothelial Cell Implantation
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About 200 µl of fibrinogen (10 mg/ml) and thrombin (10 U/ml; Thermo Fisher, Massachusetts, USA) were mixed in a centrifuge tube at room temperature. Then 2 × 104 LECs were suspended in the fibrinogen-thrombin mixture before gelation. The fibrin scaffolds were freshly prepared directly before the animal surgery and implantation.
4
Recombinant Protein Purification and Antibody Generation
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The N-001 was prepared as described previously24 (link),25 (link). In brief, plasmid encoded constructs for C2I and C2II-C1 were expressed in E. coli BL21. E. coli was cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C and were induced at an optical density of ~ 0.6 at 600 nm wavelength with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). A French pressure cell was used to lyse the cell paste at 690 bar. Glutathione resin (Genscript) was used for affinity purification. GST fusion tags were removed using thrombin (Thermo Fisher). C2II-C1 was further activated using trypsin by incubation at 37 °C for 30 min at a 1:5 enzyme to substrate ratio as previously described45 (link). A custom-made polyclonal antibody targeting the N-001 enzymatic component C2I, was prepared in rabbits. The other antibodies used were goat anti-mouse IgG (H/L) polyclonal antibody (BioRad, Cat no. STAR207P) and goat anti-rabbit IgG (H/L): HRP (BioRad, Cat no. STAR124P). Thermo Scientific Pierce TMB substrate was used for ELISAs.
5
Fabrication of Collagen-Fibrin Hydrogel
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All of the preparation of the collagen hydrogel was performed in an icebox. We first mixed 50 μL 10 × phosphate buffered saline solution (PBS) with 450 μL 3 mg/mL type I human collagen solution (VitroCol®, Advanced BioMatrix, USA), and then we adjusted the pH to 7.0 with 5 μL 1 mol/L NaOH solution. Human fibrinogen (EMD Millipore Corporation, USA) was dissolved in PBS at a concentration of 6 mg/mL at 37 °C. Thrombin (100 units/mL, Thermo Fisher Scientific) was diluted to 1 unit/mL in the medium with suspended hADMSCs (2 × 106/mL). For hydrogel formation, 100 μL of human collagen, 100 μL of Human fibrinogen, and 100 μL of hADMSCs suspension with 1 unit/mL Thrombin were mixed. The final concentrations of human collagen and fibrin were 0.9 mg/mL and 2 mg/mL, respectively.
6
Platelets PS Exposure, Mitochondria Analysis
7
Recombinant Antigen Expression and Purification
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The VHH domain was expressed and purified as described (Bloch et al., 2021 (link)). For other antigens, the gene encoding each antigen was cloned into a mammalian expression vector by bacterial homologous recombination (Fels et al., 2020 ). To facilitate purification, a thrombin cleavage site followed by a hexa‐histidine tag was fused to the C‐terminus of EPHA2‐FN2, EPHA2‐CRD, and antigen‐A, whereas a papain cleavage site followed by an Fc‐tag was fused to the C‐terminus of antigen‐B. Antigen expression vectors were transfected in mammalian cell culture using the Expi293 expression system (ThermoFisher # A14635), as described above. Cell culture expressing EPHA2‐CRD or antigen‐B was supplemented with 5 mM Kifunensine (MedChemExpress) to inhibit mannosidase I activity. EPHA2‐FN2, EPHA2‐CRD, and antigen‐A were purified using His 60 Ni Superflow resin (Takara), while antigen‐B was purified with rProteinA Sepharose (GE Healthcare). Affinity tags were cleaved by incubation with either thrombin (Merck) or papain (Thermo Scientific). Antigen‐B was further purified from Fc using rProteinA Sepharose (GE Healthcare), followed by deglycosylation with endoH (New England Biolabs). Purified protein was buffer exchanged into 20 mM HEPES pH 7.5, 100 mM NaCl, and clarified by centrifugation.
8
Fibrin Hydrogel Formation and Cell Encapsulation
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Human plasma derived fibrinogen and thrombin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Both materials were reconstituted in, Ca 2+ and Mg 2+ free, phosphate buffered saline (PBS) (Invitrogen) at 50 mg/ml and 100 U/ml, respectively, and further diluted with the same buffer before hydrogel formation. Fibrin hydrogels of 2 and 4 mg/ml were prepared using 2.5 U/ml of thrombin and cast in tissue culture plates (Thermo Fisher Scientific). Transfection and characterization assays were performed with 100 µl gels cast in 96 well plates. In differentiation assays, 200 µl gels were cast in 48 well plates. For 100 µl gels, 20 µl of the fibrinogen solution (20 or 40 mg/ml) were added to the bottom of the plate. Then, 10 µl of the cell suspension mixed with 50 µl of OptiMEM (control gels), the cell suspension mixed with 50 µl of 3DFectIN® complexes (gene-activated gels), or 60 µl of OptiMEM (blank gels) were thoroughly blended with fibrinogen by pipetting up-down. After this, 20 μl of the thrombin solution (12.5 U/ml) were quickly mixed with the previous hydrogel blend (1.5x10 6 cells/ml of gel) avoiding the formation of bubbles. Plates were placed in the cell incubator at 37°C for 1 h to allow gelation. Finally, 200 µl of culture medium were added on top of each gel. For 200 µl gels, the same procedure was followed but quantities were doubled.
9
Monoclonal Antibody-Induced Endothelial Stimulation
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Mouse IgG2a anti-human monoclonal HLA class I antibody clone W6/32 (Sigma-Aldrich, St Louis, MO), mouse IgG2a anti-human monoclonal HLA-DR, DP, DQ clone Tü39 (BD Biosciences, San Diego, CA), and clone WR18 (ThermoFisher Scientific, Rockford, IL) at 1 or 2 μg/mL were used to stimulate endothelial cells for 10 minutes or 1 hour. The concentrations of anti-HLA antibodies used are the expected physiological concentrations in human blood, as measured after the purification of DSAs from patients using specific immunoaffinity columns. 17 Stimulation with histamine (Prepotech, Rocky Hill, NJ) at 1.0 mmol/L for 10 minutes or thrombin at 1 U/mL (ThermoFisher Scientific) for 1 hour was used as a positive control. Tacrolimus was obtained from Enzo Life Sciences Inc. (Farmingdale, NY). Assessment of cell viability and the methods used to test cyclosporine, prednisone, and mycophenolic acid are described as Supplemental Material.
10
Cardiac Tissue Engineering Protocols
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The suppliers of the materials used in these experiments are as follows: Sigma‐Aldrich supplied isoprenaline (I6504); noradrenaline (A7257); milrinone (M4659); tadalfil (Y0001417); Pluronic F127, (P2443); fibrinogen, (F8630); insulin, (I9278); aprotinin, (A1153). R&D Systems supplied cilostamide (091510) and rolipram (90510) and Gibco supplied penicillin/streptomycin (15140) and Geltrex (A1413302). Other sources of compounds were: EDTA: Roth (8043.2); collagenase II, Worthington (LS004176); anti‐cardiac troponin T‐FITC, recombinant human IgG1, clone REA400, 1:10, Miltenyi Biotec (130–106‐687); agarose, Invitrogen (15510–027); thrombin, Peprotech (100–21); horse serum, Life technologies (26050088); DMEM, Biochrom (F0415).
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