After instrumentation, neuromuscular blockade was achieved by slow intravenous administration of 2 mg/kg of vecuronium bromide (Vecuronium Bromide, Hospira, Lake Forest, IL, USA). CA was induced via asphyxiation by switching off the ventilator. CA was defined as a mean arterial pressure (MAP) of less than 20 mmHg. After 12 min of asphyxia, mechanical ventilation was restarted and CPB was started. Activated coagulation time (ACT) was kept above 250 s by administering heparin (Heparin, SAGENT Pharmaceuticals, Schaumburg, IL, USA). The CPB circuit was primed using a total volume of 21.1 mL priming solution composed of 10 mL of Plasma-Lyte A (Baxter, Deerfield, IL, USA), 10 mL of 6 % Hetastarch (Hospira, Lake Forest, IL, USA), 0.8 mL of 0.406 mEq/mL magnesium sulfate (Magnesium Sulfate, APP Pharmaceuticals, Schaumburg, IL, USA), and 0.3 mL of 3.3 mmol/mL THAM Solution (XVIVO Perfusion AB, Göteborg, Sweden). The hemofiltration circuit was primed with the same solution using a priming volume of 6.9 mL. CPB was manipulated for a total of 30 min in all animals. Vasopressors or inotropic agents were not injected. At the end of CPB operation, the remaining blood in the circuit was collected and slowly reinfused into the animal.
Hetastarch
Manufactured by Pfizer
Sourced in United States
Hetastarch is a sterile, nonpyrogenic, isotonic solution used as a plasma volume expander. It is a hydroxyethyl starch derivative with an average molecular weight of 670,000 Daltons and a degree of molar substitution of 0.75. Hetastarch is composed of hydroxyethyl starch and sodium chloride in water for injection.
Automatically generated - may contain errors
Lab products found in correlation
Plasma lyte a, by Baxter (1 mentions)
Thrombopoietin, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Cryostor cs10, by BioLife Solutions (1 mentions)
Direct cd34 progenitor cell isolation kit, by Miltenyi Biotec (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Flt3 ligand, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Pfizer (1 mentions)
Minimacs columns, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
3 protocols using hetastarch
1
Asphyxia-Induced Cardiac Arrest Model
2
Placental CD34+ Cells for PNK Generation
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Placental CD34+ cells were acquired from healthy donors under fully informed consent. With donor eligibility documentation, tissues were qualified using a series of tests including serology and bacteriology (Lifebank USA). Blood was isolated from healthy donor tissues and processed by red blood cell depletion using Hetastarch (Hospira). The resulting cells were then magnetically labeled using Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). CD34+ cells were positively selected using AutoMACS Cell Separator following manufacturer’s protocol. Placental CD34+ cells were then cryopreserved in CryoStor CS10 (Biolife Solutions) and stored in liquid nitrogen before use.
For PNK culture, placental CD34+ cells were thawed and cultivated following a three-stage process in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 (Thermo Fisher Scientific), for 35 days to generate PNK cells. Nucleofection of CRISPR reagents was performed at day 5-7 of culture. Cell count and passage were performed every 2–3 days and cell expansion was recorded. At the end of the culture, cell phenotype was evaluated by flow cytometry to confirm that the cells expressed typical NK receptors and cytolytic markers.
For PNK culture, placental CD34+ cells were thawed and cultivated following a three-stage process in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2 (Thermo Fisher Scientific), for 35 days to generate PNK cells. Nucleofection of CRISPR reagents was performed at day 5-7 of culture. Cell count and passage were performed every 2–3 days and cell expansion was recorded. At the end of the culture, cell phenotype was evaluated by flow cytometry to confirm that the cells expressed typical NK receptors and cytolytic markers.
3
Isolation of CD34+ Cells from Umbilical Cord Blood
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Umbilical cord blood was collected from normal full-term delivery after obtaining informed consent from the mothers as a donation for banking, and only cord blood samples not appropriate for banking (< 100 ml) were used in our experiments. We mixed cord blood with 6% hetastarch in 0.9% sodium chloride (Hospira, USA) at a ratio of 4:1 and let it stand for approximately 30 min to allow most of the red cells to form a sediment. Cells in the supernatant was laid onto Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Pittsburgh, USA) and centrifuged to collect mononuclear cells (MNCs) by depleting the platelets, plasma, and residual red cells. Enrichment of CD34+ cells was performed with two runs of immunomagnetic selection on MiniMACS columns (Miltenyi Biotec, Gladbach, Germany) in accordance with the manufacturer’s instructions.
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