T4648

Manufactured by Merck Group

Sourced in United States

The T4648 is a laboratory equipment designed for specific scientific applications. It serves a core function within the research and development processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information on the intended use of this product is not available.

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Lab products found in correlation

12 protocols using t4648

Fabrication of Wnt5a-Loaded Chitosan Nerve Conduit

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The chitosan nerve conduit (CNC) was fabricated based on a previous protocol (National Invention Patent No. ZL01136314.2).¹⁷ We mixed 100 UI/mL thrombin (T4648, Sigma‐Aldrich) and 0.8% fibrinogen (f8630, Sigma‐Aldrich, T4648, Sigma‐Aldrich) solutions at a ratio of 1:4 to form the fibrin hydrogel.¹⁵, ¹⁸ We created a Wnt5a‐loaded fibrin hydrogel by mixing the Wnt5a solution (7815‐NG, R&D Systems) in thrombin and fibrinogen solutions, respectively, before the fibrin hydrogel was synthesized. As suggested,⁹ the final concentration of Wnt5a was 100 ng/mL.

Liu Y., Chen X., Zhou L., Rao F., Zhang D, & Wang Y. (2021). A nerve conduit filled with Wnt5a‐loaded fibrin hydrogels promotes peripheral nerve regeneration. CNS Neuroscience & Therapeutics, 28(1), 145-157.

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Hydrogel Composition Fabrication

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The following components were used for the fabrication of different hydrogel compositions: fibrinogen type I-S (60–85% protein, ~10% sodium citrate, and ~15% sodium chloride, Sigma-Aldrich F8630, Arklow, Ireland), thrombin from bovine plasma (Sigma-Aldrich, T4648, Arklow, Ireland), hyaluronic acid sodium salt from Streptococcus equi (MW = 1.5 − 1.8 × 10⁶ Da, Sigma-Aldrich 53747, Arklow, Ireland), and collagen type I, rat tail high concentration (Corning™ 354249, Corning, NY, USA).

Gansau J, & Buckley C.T. (2018). Incorporation of Collagen and Hyaluronic Acid to Enhance the Bioactivity of Fibrin-Based Hydrogels for Nucleus Pulposus Regeneration. Journal of Functional Biomaterials, 9(3), 43.

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Quantifying Thrombin Activity in Tissue

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Thrombin activity was assessed using a fluorogenic thrombin substrate (Bachem I-1560, excitation 360 nm; emission 465 nm), as previously described [18 (link)]. Human biopsies were thawed and washed 3 times for 5 min in Tris-buffer (contains in mM: 150 NaCl, 1 CaCl₂, 50 Tris-HCl: pH 8.0) on ice. Human and rodent biopsies were placed in a black 96-well microplate (Nunc, Roskilde, Denmark) containing Tris-buffer. The plate was incubated at 37 °C for 30 min before the addition of the substrate. To eliminate the effect of abundant endopeptidases in the assay, endopeptidase inhibitors (0.1 mg/mL, bestatin hydrochloride—B8385, Sigma; 200 μM prolyl endopeptidase inhibitor II, 537011, Merck Millipore) were added to the substrate. Known bovine thrombin concentrations (T-4648, Sigma) were used to create a calibration curve for each experiment. The specific thrombin inhibitor SIXAC (100 nM, American Peptide Company, Sunnyvale, CA, USA) [19 (link)] was added to chosen wells to assess the specificity of the assay. The cleavage of the substrate was measured using a microplate reader (Tecan; infinite 200; Männedorf, Switzerland). Each biopsy was weighed, and the measured thrombin activity was normalized to tissue weight. The results are presented as mU thrombin activity/mg of tissue or relative to the control.

Golderman V., Berkowitz S., Guly Gofrit S., Gera O., Aharoni S.A., Zohar D.N., Keren D., Dori A., Chapman J, & Shavit-Stein E. (2022). Thrombin Activity in Rodent and Human Skin: Modified by Inflammation and Correlates with Innervation. Biomedicines, 10(6), 1461.

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Vascularized Hydrogel Micro-Tissue Engineering

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Fibrinogen solution was made by dissolving bovine fibrinogen (10 mg/ml, F 8630, Sigma-Aldrich) in Dulbecco’s phosphate-buffered saline (DPBS, Hyclone) and filter sterilized (0.22 μm pore). Then, Mixing with aprotinin (0.15 U/mL, Sigma-Aldrich). HUVECs and LFs, which are detached from the cell culture dishes by treating 0.25% Trypsin-EDTA (Hyclone), were centrifuged and suspended at concentration of 6.7 million cells per ml in EGM-2 medium. The cell suspensions are mixed with the fibrinogen solution at a ratio of 3:1 to yield a final concentration of HUVECs and LFs as 5 million cells per ml, respectively. The mixtures with thrombin (0.5 U/ml, T4648, Sigma-Aldrich) were injected into the center hydrogel micro-channel and side micro-hydrogel channel. After 5 minutes at room temperature, the gel mixtures formed structures and the upper reservoirs in each device were filled with culture medium (EGM-2) and aspirated gently at the lower reservoirs to make the hydrophobic medium micro-channel. Following filling evenly rest of reservoirs with the medium, the devices were incubated at 37 °C and 5% CO₂. The culture medium was changed into fresh EGM-2 culture medium every 48 hours.

Bang S., Lee S.R., Ko J., Son K., Tahk D., Ahn J., Im C, & Jeon N.L. (2017). A Low Permeability Microfluidic Blood-Brain Barrier Platform with Direct Contact between Perfusable Vascular Network and Astrocytes. Scientific Reports, 7, 8083.

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Engineered Muscle Bundles Formation

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Engineered muscle bundles were formed within polydimethylsiloxane (PDMS) molds containing semi-cylindrical wells (1.25 cm long, 3 mm diameter) cast from 3D-machined Teflon masters. PDMS molds were coated with 0.2% (w/v) pluronic (P3000MP, Invitrogen) to prevent hydrogel adhesion and two Velcro felts (2mm x 4mm) were pinned at ends of the wells to anchor the hydrogel. The cell/hydrogel mixture (10 million cells/mL, 2x growth medium, 4mg/mL bovine fibrinogen (F8630, Sigma), Matrigel (20% (v/v)), and thrombin (0.2 unit/mg fibrinogen, T4648, Sigma)) was injected into the PDMS wells and polymerized at 37°C for 45 min before addition of growth medium. The formed bundles were either cultured at static or dynamic (rocked at 24 Hz, −30° to +30° tilt) conditions for 2 weeks. After 4 days of culture, growth medium was replaced by differentiation medium (DMEM, 3% (v/v) horse serum, 50 unit/mL penicillin G, 50 ug/mL streptomycin, 5 ug/mL gentamicin) to promote differentiation of the myogenic cells into myofibers. Degradation of fibrin was inhibited by 1 mg/mL aminocaproic acid (A2504, Sigma) added to culture media. Cell-mediated hydrogel compaction generated passive tension between anchored hydrogel ends resulting in uniaxial cell alignment [13 (link)].

Juhas M, & Bursac N. (2014). Roles of Adherent Myogenic Cells and Dynamic Culture in Engineered Muscle Function and Maintenance of Satellite Cells. Biomaterials, 35(35), 9438-9446.

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Thrombin-Induced Neuronal Injury

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Neurons were divided into four groups: nontreated group: neurons were treated with NB as a control; thrombin group: neurons were incubated with 100 U/ml thrombin; SP600125 (Beyotime, China) or dimethyl sulfoxide group: neurons were pretreated with 10 μM SP600125 or dimethyl sulfoxide for 2 h and then incubated with 100 U/ml thrombin (T-4648; Sigma); EGCG group: neurons were pretreated with 25 μM EGCG for 24 h and then incubated with 100 U/ml thrombin. All groups were incubated for 10 days in NB medium and treated with 100 U/ml thrombin for 48 h.

He Q., Bao L., Zimering J., Zan K., Zhang Z., Shi H., Zu J., Yang X., Hua F., Ye X, & Cui G. (2015). The protective role of (−)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation. Neuroreport, 26(7), 416-423.

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Fabrication of Fibrin Hydrogels

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To create the fibrin hydrogels two different solutions were prepared. The solution A, which contains 30% (v/v) thrombin from bovine plasma (T4648 40 U/mL, Sigma Aldrich, United States), 40% (v/v) Tris-buffered saline (TBS) (pH 7.4, Sigma Aldrich, United States) and 30% (v/v) 50 mM calcium chloride dihydrate (Sigma Aldrich, United States) in TBS; and the solution B, which contains a 10 mg/ml solution of fibrinogen from human plasma (Calbiochem 341,576, Sigma Aldrich, United States) in TBS. The mixture of both solutions in a 1:1 volume ratio using a mixing nozzle (Automix-Tips, DMG, Germany) gives the corresponding fibrin hydrogels, after a minimum incubation time of 30 min at r. t.

González-Pérez F., Acosta S., Rütten S., Emonts C., Kopp A., Henke H.W., Bruners P., Gries T., Rodríguez-Cabello J.C., Jockenhoevel S, & Fernández-Colino A. (2022). Biohybrid elastin-like venous valve with potential for in situ tissue engineering. Frontiers in Bioengineering and Biotechnology, 10, 988533.

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Co-seeding of mFBs and THP-1 Cells in Fibrin-based Scaffolds

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After cell expansion, the mFBs (passage 6) and THP-1 cells (passage 10 after thawing) were seeded in the pre-wetted scaffolds using fibrin as a cell carrier^{42 (link)}. Prior to seeding, the THP-1 cells were primed for 15 min in 50 ng ml⁻¹ phorbol 12-myristate 13-acetate (Sigma, P8139)-enriched culture medium to stimulate macrophage differentiation. To co-seed the cells into the porous scaffold, cells (15 ⋅ 10⁶ mFBs cm⁻³ and 30 ⋅ 10⁶ THP-1 cells cm⁻³) were added to a mixture of bovine fibrinogen (10 mg ml⁻¹; Sigma, F8630) and bovine thrombin (10 IU ml⁻¹; Sigma, T4648), and carefully pipetted in two steps onto two opposing sides of the scaffold. Directly after pipetting the cell suspension, the cell-seeded scaffold was manually rotated until the suspension was completely absorbed by the scaffold^{21 (link),22}. To complete fibrin polymerization, the cell-seeded constructs were transferred to a 15 ml tube and kept in an incubator (37 °C, 5% CO₂) for 30 min, after which the tubes were filled with 8 ml of complete medium. To allow for cell adhesion to the scaffold fibers, the constructs were statically cultured in these tubes for 3 days prior to exposing the constructs to the different hemodynamic loading conditions (Fig. 2d).

van Haaften E.E., Quicken S., Huberts W., Bouten C.V, & Kurniawan N.A. (2021). Computationally guided in-vitro vascular growth model reveals causal link between flow oscillations and disorganized neotissue. Communications Biology, 4, 546.

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Purification and Binding of GST-Fusion Proteins

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GST fusion proteins were expressed in E. coli strain BL21 and purified using Glutathione-Sepharose 4B beads (71024800-GE, GE Healthcare). The purified GST-fusion proteins GST-zEtv2-(236-345) and GST-zScl were treated with thrombin (1:1000; T4648, Sigma) to cleave their GST tags. For in vitro binding assays, GST-AKT1 proteins were immobilized using Glutathione-Sepharose 4B beads and incubated with either purified zEtv2-(236-345) or zScl at 4°C for 3 h. After washing, bound proteins were separated with SDS-PAGE and analyzed using Coomassie Blue staining.

Liu J., Zhang M., Dong H., Liu J., Mao A., Ning G., Cao Y., Zhang Y, & Wang Q. (2022). Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis. Development (Cambridge, England), 149(23), dev200754.

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Fibrin Gel Encapsulation of Regenerative Cells

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RJCFs were embedded in fibrin gels at a cell density of 1 million cells/ml. Gels were produced as described previously (Sander et al., 2011 (link); El-Hattab et al., 2020 (link)) by combining cells with dissolved fibrinogen (F-8630, Sigma, St. Louis, MO), 20 mM HEPES buffered saline (H-0887, Sigma, St. Louis, MO), calcium chloride (Avantor, Center Valley, PA), and dissolved thrombin (T-4648, Sigma, St. Louis, MO). 2 ml of this solution were then added to the cavities of each bioreactor and allowed to polymerize at room temperature for 10 min before being transferred to the incubator for an additional 30 min of polymerization. After polymerization, the U-shaped stabilization bar was carefully removed, the gel was gently released from the side walls, and 25 ml of DMEM supplemented with 1 ng/ml of TGF-β1(PeproTech, Inc., Cranbury, NJ) and 50 μg/ml of ascorbic acid was added to the culture dishes. The bioreactors were maintained for up to 6 days. No media changes were made to prevent disruption of the gel-wire configuration during imaging.

Scholp A.J., Jensen J., Chinnathambi S., Atluri K., Mendenhall A., Fowler T., Salem A.K., Martin J.A, & Sander E.A. (2022). Force-Bioreactor for Assessing Pharmacological Therapies for Mechanobiological Targets. Frontiers in Bioengineering and Biotechnology, 10, 907611.

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