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13 protocols using streptokinase

1

Catheter Implantation and Maintenance Protocol

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Implantations of intravenous catheters with back mounts were performed under anesthesia with a cocktail containing 8.6 mg/kg of xylazine, 1.5 mg/kg of acepromazine, and 43 mg/kg of ketamine in bacteriostatic saline and allowed to recover for 5–7 days.24 (link),28 (link),41 (link),66 (link) Catheter patency was maintained by daily flushes with a solution of 0.1 mL bacteriostatic saline containing heparin sodium (10 U/mL; American Pharmaceutical Partners, East Schaumburg, IL), streptokinase (0.67 mg/mL; Sigma Chemical), and ticarcillin disodium (66.67 mg/mL; Research Products International, Mt. Prospect, IL) immediately following daily oxycodone self-administration sessions. Proper catheter function was verified periodically throughout experiments by intravenous administration of 10 mg/kg of methohexital sodium (Monarch Pharmaceuticals Inc., Bristol, TN), a dose sufficient to briefly anesthetize the animal only when administered intravenously. All rats were allowed 5–7 days of recovery after surgery before initiation of self-administration training.
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2

Isolation of Trophoblastic and Amniotic Cells

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The ACM was flattened in Trypsin-EDTA diluted with PBS (Trypsin–EDTA, 0.1% w/v, Sigma-Aldrich, USA) for 20–30 min at 37 °C. After washing the membrane in Dulbecco’s modified Eagle’s medium (DMEM) culture medium (pH 7.4), the membrane was incubated in streptokinase (0.02% w/v, Sigma-Aldrich, USA) for 8–10 min at 37 °C and subsequently, the membrane was washed with sterile DMEM culture medium (pH 7.4) once and lastly with PBS three times.
Following these treats, trophoblastic cells of the chorionic side of the membrane were gently scraped out in PBS by a plastic scraper without detaching the amniotic membrane from the chorionic membrane or rupture of the membrane for 10–15 min at 25 °C. Epithelial cells of the amniotic side of the membrane were scraped out gently by scraping with the same strength in PBS for 10–15 min. Finally, the scaffold was washed in PBS three times.
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3

Cocaine Self-Administration in Rats

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Rats (n = 156) were implanted with intravenous catheters with back mounts under anesthesia with a cocktail containing 8.6 mg/kg of xylazine, 1.5 mg/kg of acepromazine, and 43 mg/kg of ketamine in bacteriostatic saline and allowed to recover for 5–7 days before initiation of self-administration training (Anastasio et al., 2014a (link), 2014b (link), 2013; Neelakantan et al., 2017 (link); Sholler et al., 2019 ). Catheter patency was maintained by daily flushes with a solution of 0.1 ml bacteriostatic saline containing heparin sodium (10 U/ml; American Pharmaceutical Partners, East Schaumburg, IL), streptokinase (0.67 mg/ml; Sigma Chemical), and ticarcillin disodium (66.67 mg/ml; Research Products International, Mt. Prospect, IL) immediately following daily cocaine self-administration sessions.
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4

Rat Catheter Implantation and Maintenance

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Rats were anesthetized with a cocktail of xylazine (8.6 mg/kg), acepromazine (1.5 mg/kg), and ketamine (43 mg/kg), and indwelling catheters were implanted into the right jugular vein. Catheters were secured to a plastic back mount and surgical mesh, as previously described (Cunningham et al., 2011 (link); Neelakantan et al., 2017 (link); Sholler et al., 2019 (link)), and rats were allowed 7 days of recovery after surgery. Catheter patency was maintained with a bacteriostatic saline infusion (0.1 mL) containing streptokinase (0.67 mg/mL; Sigma Chemical, St. Louis, MO, USA), heparin sodium (10 U/mL; American Pharmaceutical Partners, East Schaumburg, IL, USA), and ticarcillin disodium (66.67 mg/mL; Research Products International, Mt. Prospect, IL, USA) administered immediately after daily self-administration sessions.
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5

Biochemical Analyses of Natural Compounds

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Phosphate‐buffered saline (PBS), 4‐Nitrophenyl β‐D‐glucopyranoside (pNPG), 3,5‐dinitro salicylic acid (DNS), caryophyllene, bovine serum albumin (BSA), α‐tocopherol, α‐amylase, α‐glucosidae, streptokinase, aspirin, 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH), and acarbose were purchased from Sigma‐Aldrich, Singapore. All other reagents and solvents were of analytical grade.
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6

Murine Immune Cell Profiling Protocol

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Pac Blue anti-CD45 mAb, allophycocyanin anti-ICAM2 mAb, allophycocyanin/Cy7 anti-CD11b mAb, PE anti-Ly6G mAb, PerCP/Cy5.5 anti-Ly6C mAb, PE/Cy7 anti-F4/80 mAb, and anti-ICAM2 mAb were all purchased from BioLegend. Anti-S100A9 polyclonal antibody was purchased from R&D Systems (AF2065-SP). Anti-Gata6 monoclonal antibody was purchased from Cell Signaling Technology (5851). Anti-fibrin(ogen) polyclonal antibody was purchased from Agilent Dako (A0080). The Alexa Fluor 594–conjugated heat-killed bacterial particles were purchased from Thermo Fisher Scientific (E23370). Clodronate-loaded liposome was purchased from ClodronateLiposomes.org. Recombinant hirudin was purchased from Aniara Diagnostica (ARE120A). Zymosan was purchased from Sigma-Aldrich (Z4250). GFP E. coli was purchased from ATCC (ATCC 25922GFP). Human FV–deficient plasma was purchased from Haematologic Technologies. Rabbit thromboplastin (44213), Liberase, hyaluronidase, DNase I, streptokinase, and collagenase D were purchased from Sigma-Aldrich. Plasminogen was purchased from Lee Biosolutions.
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7

Blood Collection and Clotting Analysis Protocol

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Human venous blood was collected from healthy consenting volunteers using butterfly needles (BD Vacutainer, BD Safety-Lok, Cat # 367283BD, Franklin Lakes, NJ) and stored in 4.5 mL citrated tubes (BD Vacutainer, Buffered Cit. Na., Cat # 366415), and kept at 22 °C Blood sample procurement protocols were approved by IRB of the University of North Carolina at Chapel Hill (IRB # 12-1592). Blood samples were used within 24h of collection and run as split specimens on the ASAP system and a TEG 5000 analyzer (Heomentics, Inc). For the dilution experiments, isotonic saline (0.90% w/v NaCl) was added to the blood and mixed by gently inverting the tube. Streptokinase, purchased from Sigma-Aldrich (product # S3134-10KU), was diluted to a final concentration of 9.6 U/ml for the high dose lysis experiment (Figure 4, blue circles) and 1.2 U/mL for the low dose lysis experiment (Figure 4, green squares). Immediately prior to data collection, all specimens were re-calcified by adding 1 μL of 0.2M CaCl per 17 μL of specimen volume. A 20 μL droplet of blood was pipetted onto the opening of the micro-fluidic device, and the blood wicked into the channel. The final, as tested, specimen volumes for the ASAP and TEG systems were 20 μL and 360 μL, respectively. The TEG was run according to standard operating procedures.
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8

Lymphocyte Stimulation Assay

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Isolated mononuclear cells from blood and gut biopsies were seeded at the concentration of 2×106 cells/ml with RPMI media+10% heat inactivated fetal bovine serum (FBS) and cultured overnight at 37°C and 5% CO2 in presence of medium alone; phorbol myristyl acetate (PMA) (3 ng/ml Sigma Aldrich, Milan Italy) and ionomycin (1 µM Sigma Aldrich, Milan, Italy); Streptokinase (SK, 10 µg/ml, Sigma Aldrich Milan, Italy); or Candida albicans mannoprotein antigen (Ca, 10 µl/ml kindly provided by Dr A. Cassone, Istituto Superiore di Sanità, Rome, Italy). Brefeldine A (BFA, Sigma Aldrich, Milan, Italy) was added to all culture conditions. Cells were collected, washed and stained for T cell phenotype.
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9

Reagents and Suppliers for Cell Culture

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Aldosterone, amphotericin B, glucose, heparin, L-glutamine, mannitol, penicillin, RU486, streptokinase, streptomycin, streptozotocin, trypsin, and trypan blue were purchased from Sigma-Aldrich (Poznan, Poland). EPL (Inspra; Pfizer, Warsaw, Poland), low-serum growth supplement (LSGS; Cascade Biologics, Portland, OR, USA), Medium 200 (M200; Cascade Biologics, Portland, OR, USA), and pentobarbitone sodium (Morbital; Biowet, Pulawy, Poland) were used. EDTA, ethanol, gum arabic, natrium chloride, magnesium chloride, and trisodium citrate were purchased from Polish Chemical Reagents (Gliwice, Poland).
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10

Coagulation and Fibrinolytic Assays

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Coagulation proteins were purchased from Calbiochem, Germany. Human thrombin, prothrombin, human fibrinogen, and extracellular matrix (ECM) proteins like type-IV collagen, laminin, and fibronectin were purchased from Sigma Aldrich, USA. All commercially available drugs, like tissue plasminogen activator, streptokinase and plasmin were purchased from Sigma Aldrich, USA. Nattokinase was purchased from Healthy Origins, Pittsburg, USA. PT and APTT kits were purchased from Tulip diagnostics, Mumbai. LDL, HDL, and triglycerides assay kits were purchased from Diatek Healthcare Pvt. Ltd., Kolkata, India. The cholesterol assay kit was purchased from Sirus Biocare Pvt. Ltd., Kolkata, India, the fibrinogen assay kit was purchased from R2 Diagnostics, USA, and the immunoglobulin EIA kits were obtained from Thermo Fisher Scientific, USA. All other reagents were of analytical grade and purchased from Sigma Aldrich, USA.
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