Green fluorescent protein (GFP) expressing P. falciparum (D10-PfPHG) (Wilson et al., 2010 (link)) and P. knowlesi (PkYHI) parasites (Lim et al., 2013 (link)) were cultured in human O+ red blood cells (RBCs) (Australian Red Cross Blood Service) in RPMI-HEPES culture medium (Thermo Fisher Scientific) supplemented with 0.5% v/v Albumax (Gibco), 52 μM gentamycin (Gibco), 367 μM hypoxanthine (Sigma-Aldrich), and 2 mM sodium bicarbonate (Thermo Fisher Scientific), adjusted to a pH of 7.2-7.4. Cultures were grown in sealed containers with 1% O2, 5% CO2 and 94% N2 (BOC Gases) at 37°C (Trager and Jensen, 1976 (link)). Synchronization of D10-PfPHG parasites for growth inhibition assays was achieved using heparin sodium (Pfizer) as previously described (Boyle et al., 2010 (link); Wilson et al., 2013 (link)). PkYH1 parasites were passaged over a gradient of 70% Percoll (Sigma-Aldrich) to purify late stage schizonts which were then allowed to rupture for ~4 hrs in the presence of fresh RBCs prior to ring-stage treatment with 5% w/v sorbitol (Sigma-Aldrich), enabling effective synchronisation of 0-4 hrs old rings.
Heparin sodium
Manufactured by Pfizer
Sourced in Australia, United States
Heparin sodium is a laboratory product that serves as an anticoagulant. It is used to prevent the formation of blood clots in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Evans blue dye, by Merck Group (2 mentions)
Sorbitol, by Merck Group (1 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Albumax, by Thermo Fisher Scientific (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Normal saline, by Pfizer (1 mentions)
Lidocaine hydrochloride, by Merck Group (1 mentions)
Cell titre glo kit, by Promega (1 mentions)
Flufenamic acid, by Merck Group (1 mentions)
Soybean lecithin, by Merck Group (1 mentions)
Aerosil 300 pharma, by Evonik (1 mentions)
Glacial acetic acid, by Chem-Supply (1 mentions)
Milli q water, by Merck Group (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Trimethoprim, by Virbac (1 mentions)
12 protocols using heparin sodium
1
Culturing Fluorescent Malaria Parasites
2
Quantitative Analysis of Lidocaine in Biological Samples
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Reference standards of lidocaine hydrochloride and monoethylglycinexylidide (≥95%) were purchased from Sigma Aldrich, Auckland, New Zealand. lidocaine hydrochloride for injection was purchased from Ethical agents Ltd., Auckland, New Zealand. Acetonitrile, methanol, water, and formic acid were mass spectrometry grade and were purchased from Fisher Scientific, Auckland, New Zealand. Heparin sodium and normal saline were purchased from Pfizer New Zealand Limited, Auckland, New Zealand, and Baxter Healthcare Pty Ltd., Old Toongabbie, NSW, Australia, respectively. Artificial colostrum and milk replacer were purchased from Farmlands Co-Operative Society Ltd., Palmerston North, New Zealand.
3
Heparin Inhibits SARS-CoV-2 Infection
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Vero and ALI cells were pretreated with either 125 µg or 250 μg of heparin sodium (Pfizer, New York, NY, USA) for 30 min prior to infection with SARS-CoV-2 as detailed above. Following infection (unless indicated otherwise), cells were cultured in the presence of either 125 μg/mL or 250 μg/mL of heparin throughout the experiment. For ALI nasal epithelium, cells were treated with heparin on both apical and basolateral surfaces, with heparin replaced daily on the apical surface and every 2 days on the basolateral surface. In some experiments, the virus was incubated with heparin (250 μg) for 1 h at room temperature prior to infection, as described above. The virus/heparin solution was removed from the cells by washing with either serum-free MEM (Vero) or PBS (ALI cells) and cultured with or without heparin for the remainder of the experiment. Cell viability in the presence of heparin was assessed using the CellTitre-Glo kit (Promega, Madison, WI, USA) as per the manufacturer’s instructions.
4
Cord Blood Collection and Purification
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Cord blood is collected from the umbilical cord vein after the baby has been born and the cord has been clamped and cut. The vein must be swabbed with alcohol and maternal blood gently removed before the collection of the sample using an 18 Gauge SafetyGlide™ needle (Cat No. 305918, Becton Dickinson) and a 30 mL syringe. The gauge of the needle limits cell damage due to shredding. Blood is transferred into all necessary tubes through the syringe or other transfer device; pouring between tubes must not occur in order to prevent cross-contamination of tube preservatives. The majority of the sample is transferred into a room-temperature 50 mL conical tube containing 20 mL of Gibco™ RPMI 1640 Medium and 500 µL of Heparin Sodium (Cat No. AUST R 49232, Pfizer, New York, NY, USA) to support cell survival. As cord blood tends to have more red blood cell contamination than peripheral blood collections, two Lymphoprep™ density gradient steps are required.
5
Hyperinsulinemic Euglycemic Clamp in Mice
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Mice were subjected to jugular vein catheterization 5 days prior to hyperinsulinemic euglycemic clamp studies. Mice were anesthetized with isoflurane (2–3% in oxygen) while an indwelling silastic catheter was inserted into the right internal jugular vein and exteriorized through the back of the neck. The catheters were kept patent with heparin sodium (1 IU/ml, Pfizer) and sealed with a stainless steel plug. Mice were allowed 4–5 days postsurgical recovery. Food pellets were placed at the bottom of the cage to facilitate recovery. Body weight was recorded daily, and mice that had less than 5% weight loss were subsequently studied.
Hyperinsulinemic euglycemic clamps followed by 2-deoxyglucose uptake were performed on 6 h fasted, conscious, and unrestrained mice as described previously [19] (link). Insulin infusion rate for chow fed mice was 2 mU/kg/min, and for high-fat diet fed animals was 4 mU/kg/min. Soleus, extensor digitorum longus, gastrocnemius, tibialis anterior, kidney, liver, WAT (visceral, perigonadal and subcutaneous), BAT, and heart were assessed for 2[14C]DG radioactivity.
Hyperinsulinemic euglycemic clamps followed by 2-deoxyglucose uptake were performed on 6 h fasted, conscious, and unrestrained mice as described previously [19] (link). Insulin infusion rate for chow fed mice was 2 mU/kg/min, and for high-fat diet fed animals was 4 mU/kg/min. Soleus, extensor digitorum longus, gastrocnemius, tibialis anterior, kidney, liver, WAT (visceral, perigonadal and subcutaneous), BAT, and heart were assessed for 2[14C]DG radioactivity.
6
Analytical Characterization of Ibuprofen Formulation
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The drug of interest, ibuprofen (IBU) ($98%, GC), and internal standard flufenamic acid (ISTD) (analytical grade) were purchased from Sigma Aldrich (Castle Hill, Australia). Commercially available Nurofen tablets (Reckitt Benckiser, Sydney, Australia) were crushed into a uniform powder before use. The lipid, Capmul PG8 (propylene glycol caprylate), was sourced from Abitec (Columbus, OH). Glacial acetic acid (analytical grade) was purchased from Chem-Supply (Gillman, Australia). Silica nanoparticles (Aerosil 300 Pharma), with a surface area of 300 6 30 m 2 /g were donated by Evonik (Melbourne, Australia). Nanoporous silica microparticles (Parteck SLC 500), with a 9-11 mm particle size and 6 nm pore size, were donated by Merck (Bayswater, Australia). Soybean lecithin and liquid chromatography grade methanol were purchased from Merck. Heparin sodium (5000 IU/5 ml) was purchased from Pfizer (Perth, Australia). High purity Milli-Q water was acquired from a Milli-Q water purification system (Merck).
7
Detailed Surgical Protocols for Small Animal Models
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Isoflurane and pentobarbital sodium (Lethabarb®) were purchased from Pro-Vet (Victoria, Australia). Heparin sodium was purchased from Hospira (Victoria, Australia). Saline was purchased from Baxter International Inc. (New South Wales, Australia). Polyethylene cannulas (0.96 mm I.D. x 0.58 mm O.D., 0.80 mm O.D. x 0.5 mm I.D. and 0.5 mm O.D. x 0.2 mm I.D.) were purchased from Microtube Extrusions (New South Wales, Australia). All surgical tools, with one exception, were purchased from Fine Science Tools (United States): 2 x straight forceps (11150-10), 2 x haemostats (91308-12), 1 x iris scissors (91500-09), 2 x blunt forceps (11152-10). 1 x fine-tipped titanium forceps (TY-1301) was purchased from ProSciTech (Australia). All fluorescent labelled antibodies and viability dye for flow cytometry were purchased from Thermo Fisher Scientific (Victoria, Australia).
8
Hemorrhage-Induced Physiological Response in Rats
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Male Sprague Dawley rats (weight = 425 [6] g) were obtained from our institutional breeding colony, and housed in a 14/10 hour light/dark cycle with free access to food and water ad libitum. Animals were heparinized with 2,500 IU Heparin Sodium (Hospira, Lake Forest, Illinois) and anesthetized intraperitoneally with 100 mg/kg sodium thiopentone (Thiobarb, Rutherford, Australia). Anesthetic was administered as required throughout the protocol. The study was approved by our institutional Animal Ethics Committee (No. A1148) and conforms to the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition; 2013) and the Guide for Care and Use of Laboratory Animals (8th edition; 2011). As per the Australian code, the 3Rs (ie, replacement, reduction, and refinement) have been applied in this study. Hemorrhage results in the activation of multiple body systems that cannot be accurately mimicked using an alternative in vitro method (ie, replacement). Power analysis was conducted to determine the smallest number of animals necessary to achieve the study's aims (see Statistical Analysis) (ie, reduction), and depth of anesthesia was continuously monitored to ensure animal well-being (ie, refinement).
9
Cardiac Blood Extraction and Tissue Sampling
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Rats were enclosed in an anesthesia box and deeply anesthetized using 5% isoflurane mixture with air. Anesthetized animals were placed on the pad in supine position and anesthesia continued through a nose cone. The thoracic cavity was opened and ~ 9–10 ml of blood was collected by cardiac puncture, representing about two-thirds of total blood volume for a rat of this size. Heparinized syringes were used for blood collection. Immediately after withdrawal, blood was centrifuged (17,000 g, 5 min) to obtain plasma. Animals were perfused using heparin-containing saline (Heparin Sodium, 2 USP units/ml, Hospira, Lake Forest, IL) to flush the remaining blood out of tissues. Perfusion continued until no blood was detected in outgoing saline and liver was significantly discolored. Samples from liver, gluteus muscles and eyes were then collected, placed into microcentrifuge tubes and immediately frozen on dry ice. All samples were stored at − 80 °C until analysis.
10
Dual Implantation of Radio Telemeter and Vascular Access Port
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Dual implantation of a radio telemeter and vascular access port was performed on 16-week old animals under 2–2.5% isoflurane anaesthesia (Minrad Inc, Bethlehem, PA, USA) as previously described [34 (link)] with strict adherence to aseptic procedures. Analgesia (5 mg.kg−1 carprofen; Norbrook, Newry, Northern Ireland) and antibiotic (30 mg.kg−1 trimethoprim and sulphamethazine; Virbac, Carros, France) were administered subcutaneously.
Vascular Access Ports (VAP™; ROP-3H, hydromer-coated polyurethane, 3Fr; Access Technologies, Skokie, IL, USA) were primed with heparin sodium (100 IU.mL−1; Hospira Australia, Mulgrave, Australia). The VAP reservoir was secured on the back of the animal between the scapulae, with the VAP cannula tunnelled subcutaneously to the femoral vein. A radio telemeter with pressure sensitive tip (TRM53P; Telemetry Research, Millar Instruments, Houston, TX, USA) was implanted into the abdominal aorta. Animals were allowed one-week post-surgical recovery before experimentation commenced (Figure1 a). The VAP was flushed with 0.4 mL heparin sodium (100 IU.mL−1) at minimum twice-weekly to maintain patency.
Vascular Access Ports (VAP™; ROP-3H, hydromer-coated polyurethane, 3Fr; Access Technologies, Skokie, IL, USA) were primed with heparin sodium (100 IU.mL−1; Hospira Australia, Mulgrave, Australia). The VAP reservoir was secured on the back of the animal between the scapulae, with the VAP cannula tunnelled subcutaneously to the femoral vein. A radio telemeter with pressure sensitive tip (TRM53P; Telemetry Research, Millar Instruments, Houston, TX, USA) was implanted into the abdominal aorta. Animals were allowed one-week post-surgical recovery before experimentation commenced (Figure
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